Anti-IFNAR1 antibodies with reduced Fc ligand affinity

ABSTRACT

The invention provides anti-IFNAR1 antibodies with reduced affinity for Fc receptors and/or ligands and methods of making and using such antibodies.

This application is a continuation of U.S. application Ser. No. 12/866,579 filed on Nov. 9, 2010, said application Ser. No. 12/866,579 is a U.S. National Stage application of International Application No. PCT/US2009/033358, filed Feb. 6, 2009, said Application No. PCT/US2009/033358 claims benefit under 35 U.S.C. § 119(e) of the following U.S. Provisional Application Nos. 61/006,962 filed Feb. 8, 2008, 61/034,618 filed Mar. 7, 2007, and 61/049,970 filed May 2, 2008. Each of the above listed applications is incorporated by reference herein in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submitted with this application as text file entitled IFNAR-US-110-CNT_SEQUENCE_LISTING.txt created on Sep. 14, 2017, and having a size of 48,534 kilobytes.

1. FIELD OF THE INVENTION

The present invention relates to isolated antibodies and compositions specific for the interferon alpha receptor 1 (IFNAR1) with reduced affinity for Fc ligands. The invention also comprises nucleic acids encoding such antibodies, complementary nucleic acids, vectors, host cells, and methods of making and using thereof, including therapeutic compositions, formulations, administrations and devices.

2. BACKGROUND OF THE INVENTION 2.1 Interferons

Type I interferons (IFN) (IFNα, IFNβ, IFNω, IFNτ) are a family of structurally related cytokines having antiviral, antitumor and immunomodulatory effects (Hardy et al. (2001) Blood 97:473; Cutrone and Langer (2001) J. Biol. Chem. 276:17140). The human IFNα locus includes two subfamilies. The first subfamily consists of 14 non-allelic genes and 4 pseudogenes having at least 80% homology. The second subfamily, αII or omega (ω), contains 5 pseudogenes and 1 functional gene which exhibits 70% homology with the IFNα genes (Weissmann and Weber (1986) Prog. Nucl. Acid Res. Mol. Biol., 33:251-300). The subtypes of IFNα have different specific activities but they possess the same biological spectrum (Streuli et al. (1981) Proc. Natl. Acad. Sci. USA 78:2848) and have the same cellular receptor (Agnet M. et al. in “Interferon 5” Ed. I. Gresser p. 1-22, Academic Press, London 1983). Interferon alpha subtypes have been identified with the following nomenclature: IFNα 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.

The interferon β (IFNβ) is encoded by a single gene, which has approximately 50% homology with the IFNα genes.

Interferon γ, which is produced by activated lymphocytes, does not possess any homology with the alpha/beta interferons and it does not react with their receptor.

2.1.1 Interferon Receptors:

All human type I interferons bind to a cell surface receptor (IFN alpha receptor, IFNAR) consisting of two transmembrane proteins, IFNAR1 and IFNAR2 (Uze et. al. (1990) Cell 60:225; Novick et al. (1994) Cell 77:391). IFNAR1 is essential for high affinity binding and differential specificity of the IFNAR complex (Cutrone et al. 2001 J. Bio Chem 276(20):17140-8) While functional differences for each of the type I IFN subtypes have not been identified, it is thought that each may exhibit different interactions with the IFNAR receptor components leading to potentially diverse signaling outcomes (Cook et al. (1996) J. Biol. Chem. 271:13448). In particular, studies utilizing mutant forms of IFNAR1 and IFNAR2 suggested that alpha and beta interferons signal differently through the receptor by interacting differentially with respective chains (Lewerenz et al. (1998) J. Mol. Biol. 282:585).

2.1.2 Function of Interferons:

Early functional studies of type I IFNs focused on innate defense against viral infections (Haller et al. (1981) J. Exp. Med. 154:199; Lindenmann et al. (1981) Methods Enzymol. 78:181). More recent studies, however, implicate type I IFNs as potent immunoregulatory cytokines in the adaptive immune response. Specifically, type I IFNs have been shown to facilitate differentiation of naïve T cells along the Th1 pathway (Brinkmann et al. (1993) J. Exp. Med. 178:1655), to enhance antibody production (Finkelman et al. (1991) J. Exp. Med. 174:1179) and to support the functional activity and survival of memory T cells (Santini et al. (2000) J. Exp. Med. 191:1777; Tough et al. (1996) Science 272:1947).

Recent work by a number of groups suggests that IFNα may enhance the maturation or activation of dendritic cells (DCs) (Santini, et al. (2000) J. Exp. Med. 191:1777; Luft et al. (1998) J. Immunol. 161:1947; Luft et al. (2002) Int. Immunol. 14:367; Radvanyi et al. (1999) Scand. J. Immunol. 50:499). Furthermore, increased expression of type I interferons has been described in numerous autoimmune diseases (Foulis et al. (1987) Lancet 2:1423; Hooks et al. (1982) Arthritis Rheum. 25:396; Hertzog et al. (1988) Clin. Immunol. Immunopathol. 48:192; Hopkins and Meager (1988) Clin. Exp. Immunol. 73:88; Arvin and Miller (1984) Arthritis Rheum. 27:582). The most studied examples of this are insulin-dependent diabetes mellitus (IDDM) (Foulis (1987)) and systemic lupus erythematosus (SLE) (Hooks (1982)), which are associated with elevated levels of IFNα, and rheumatoid arthritis (RA) (Hertzog (1988), Hopkins and Meager (1988), Arvin and Miller (1984)), in which IFNβ may play a more significant role.

Moreover, administration of interferon α has been reported to exacerbate underlying disease in patients with psoriasis and multiple sclerosis and to induce an SLE like syndrome in patients without a previous history of autoimmune disease. Interferon α has also been shown to induce glomerulonephritis in normal mice and to accelerate the onset of the spontaneous autoimmune disease of NZB/W mice. Further, IFNα therapy has been shown in some cases to lead to undesired side effects, including fever and neurological disorders. Hence there are pathological situations in which inhibition of Type I IFN activity may be beneficial to the patient and a need exists for agents effective in inhibiting Type I IFN activity.

2.1.3 Antibody Effector Functions:

The Fc region of an antibody interacts with a number of ligands (also referred herein as “Fc ligands” which include but are not limited to agents that specifically bind to the Fc region of antibodies, such as Fc receptors and C1q) including Fc receptors and C1q, imparting an array of important functional capabilities referred to as effector functions. The Fc receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans this protein family includes FcγRI (CD64), including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32), including isoforms FcγRIIA, FcγRIIB, and FcγRIIC; and FcγRIII (CD16), including isoforms FcγRIIIA and FcγRIIB (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a membrane spanning region, and an intracellular domain that may mediate some signaling event within the cell. These receptors are expressed in a variety of immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells. Formation of the Fc/FcγR complex recruits these effector cells to sites of bound antigen, typically resulting in signaling events within the cells and important subsequent immune responses such as release of inflammation mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells. The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is referred to as antibody dependent cell-mediated phagocytosis (ADCP). In addition, an overlapping site on the Fc region of the molecule also controls the activation of a cell independent cytotoxic function mediated by complement, otherwise known as complement dependent cytotoxicity (CDC).

2.1.4 the Different Types of Human FcγR:

Human FcγRs are divided into three distinct classes: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). FcγRI is a high affinity receptor (K_(a): 10⁻⁸-10⁻⁹ M⁻¹) and binds both immune complexes and monomeric IgG molecules while the Fc receptors FcγRII and FcγRIII exhibit lower affinities (<10⁻⁷ M⁻¹ and 2-3×10⁻⁷ respectively) (Gessner J. E. et al., 1998, Annn Hematology 76:231-48). Signaling through the FcγRs is either through an immunoreceptor tyrosine-based activation motif (ITAM) or immunoreceptor tyrosine-based inhibitory motif (ITIM) for all the transmembrane receptors (Presta 2006, Adv Drug Deli Rev 58:640-656).

The 72 kDa extracellular glycoprotein FcγRI is mainly expressed on myeloid cells such as monocytes, macrophages CD4+ progenitor cells and may elicit the ADCC, endocytosis, and phagocytosis responses (Siberil et al. 2006, J Immunol Lett 106:111-118).

The 40 kDa FcγRII group of receptors (A, B and C isoforms) exhibit extracellular domains but do not contain active signal transduction domains. These receptors propagate signals through phosphorylation of a cytoplasmic tail domain (Amigorena S. et al., 1992 Science. 256:1808-12). The FcγRIIA is mainly expressed on monocytes, macrophages, neutrophils, and platelets whereas the FcγRIIC receptor has only been identified on NK cells. These two receptors have been shown to initiate ADCC, endocytosis, phagocytosis and inflammatory mediator release (Cassel et al. 1993. Mol Immunol 30:451-60). By contrast, the FcγRIIB (B1 and B2 types) receptors are expressed on B cells, Mast cells, basophils, monocytes, macrophages and dendritic cells and has been shown to downregulate the immune response triggered by the A and C isoforms.

The 50 kDa FcγRIIIA, expressed on NK cells, monocytes, macrophages and a subset of T lymphocytes where it activates ADCC, phagocytosis, endocytosis and cytokine release (Gessner et al.). The FcγRIIIB isoforms is a glycosyl-phosphatidylinositol (GPI) anchored peripheral membrane protein involved in the degranulation and the production of reactive oxygen intermediates (Salmon J. E. et al. 1995 J Clin Inves 95:2877-85).

IgG molecules also exhibit differential isotype specificity for FcγRs. IgG3 molecules bind strongly to all FcγR isoforms. IgG1, the most prevalent isoforms in the blood binds to all FcγRs albeit with a lower affinity for the FcγRIIA/B isoforms. IgG4 is an intermediate binder to FcγRI and a weak binder to FcγRIIB. Finally, IgG2 binds only weakly to one allelic form of FcγRIIA (FcγRIIA-H131) (Siberil et al. 2006, J Immunol Lett 106:111-118).

2.1.5 Complement

The complement inflammatory cascade is a part of the innate immune response and is crucial to the ability for an individual to ward off infection. Another important Fc ligand is the complement protein C1q. Fc binding to C1q mediates a process called complement dependent cytotoxicity (CDC) (reviewed in Ward et al., 1995, Ther Inmunol 2:77-94). C1q is capable of binding six antibodies, although binding to two IgGs is sufficient to activate the complement cascade. C1q forms a complex with the C1r and C1s serine proteases to form the C1 complex of the complement pathway.

2.1.6 Regions and Amino-Acid Residues of IgG Involved in FcγR Binding

The mapping of human IgG binding sites to different FcγR has been studied extensively. These studies, based on genetically altered IgG molecules have identified a short continuous stretch of amino acid residues (234-238) of the N-terminus part of the CH2 domain as being directly involved in the binding to all FcγRs. Additionally, residues 268, 297, 327 and 329 may impact binding to a subset of FcγRs. Also, multiple residues located in the CH2 and CH3 domains also contribute to FcγR binding (Canfield S M. et al., 1991 J Exp Med 173:1483-91, Chappel M S. Et al. 1991, Proc Nat Acad Sci USA 888:9036-40, Gergely J. et al. 1990 FASEB J 4:3275-83).

2.2 Antibody Therapeutic Related Toxicity

In many circumstances, the binding and stimulation of effector functions mediated by the Fc region of immunoglobulins is highly beneficial, however, in certain instances it may be more advantageous to decrease or eliminate effector function. This is particularly true for those antibodies designed to deliver a drug (e.g., toxins and isotopes) to the target cell where the Fc/FcγR mediated effector functions bring healthy immune cells into the proximity of the deadly payload, resulting in depletion of normal lymphoid tissue along with the target cells (Hutchins et al., 1995, PNAS USA 92:11980-11984; White et al., 2001, Annu Rev Med 52:125-145). In these cases the use of antibodies that poorly recruit complement or effector cells would be of tremendous benefit (see for example, Wu et al., 2000, Cell Immunol 200:16-26; Shields et al., 2001, J. Biol Chem 276:6591-6604; U.S. Pat. No. 6,194,551; U.S. Pat. No. 5,885,573 and PCT publication WO 04/029207).

In other instances, for example, where blocking the interaction of a widely expressed receptor with its cognate ligand is the objective, it would be advantageous to decrease or eliminate all antibody effector function to reduce unwanted toxicity. Also, in the instance where a therapeutic antibody exhibited promiscuous binding across a number of human tissues it would be prudent to limit the targeting of effector function to a diverse set of tissues to limit toxicity. Although there are certain subclasses of human immunoglobulins that lack specific effector functions, there are no known naturally occurring immunoglobulins that lack all effector functions. An alternate approach would be to engineer or mutate the critical residues in the Fc region that are responsible for effector function. For examples see PCT publications WO2006076594, WO 199958572, US20060134709, WO2006047350, WO2006053301, and U.S. Pat. No. 5,624,821 each of which are incorporated by reference in their entireties.

The use of monoclonal antibodies in the treatment of many disease states has been well documented. With the myriad of effector functions that an antibody can trigger, one of the requirements of antibody therapeutics is that they are targeted specifically to a target of interest. For example, but not limited to, the specificity of a target tissue is analyzed by examining the immunohistochemistry (IHC) of a tissue of interest. It is important that the therapeutic only bind to tissues that contain a target of interest. Failure to do so could result in higher toxicity of the antibody therapeutic due to inappropriate activation of effector function elicited at the non-targeted site. If the effector function could be diminished or ablated, the danger of the widespread binding of the therapeutic could be avoided. With all these considerations, there is an unmet need for antibodies with reduced or ablated affinity for at least one Fc ligand responsible for facilitating effector function. Such antibodies would be of particular benefit for use in the treatment of chronic inflammatory and autoimmune conditions.

Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.

3. BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments on the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

FIG. 1A. Nucleic acid (SEQ ID No:7) and amino acid (SEQ ID No:8) sequence alignment of 3F11 VH with the CDR regions are indicated by the overline.

FIG. 1B. Nucleic acid (SEQ ID No:9) and amino acid (SEQ ID No:10) sequence alignment of 3F11 VK with the CDR regions outlined are indicated by the overline.

FIG. 2A. Nucleic acid (SEQ ID No:17) and amino acid (SEQ ID No:18) sequence alignment of 4G5 VH with the CDR regions outlined are indicated by the overline.

FIG. 2B. Nucleic acid (SEQ ID No:19) and amino acid (SEQ ID No:20) sequence alignment of 4G5 VK with the CDR regions outlined are indicated by the overline.

FIG. 3A. Nucleic acid (SEQ ID No:27) and amino acid (SEQ ID No:28) sequence alignment of 11E2 VH with the CDR regions outlined are indicated by the overline.

FIG. 3B. Nucleic acid (SEQ ID No:29) and amino acid (SEQ ID No:30) sequence alignment of 11E2 VK with the CDR regions outlined are indicated by the overline.

FIG. 4A. Nucleic acid (SEQ ID No:37) and amino acid (SEQ ID No:38) sequence alignment of 9D4 VH with the CDR regions outlined are indicated by the overline.

FIG. 4B. Nucleic acid (SEQ ID No:39) and amino acid (SEQ ID No:40) sequence alignment of 9D4 VK with the CDR regions outlined are indicated by the overline.

FIG. 5. Amino acid sequence alignment of heavy chain constant regions for 9D4. Arrows indicate amino acid substitutions (unmodified to modified) to increase stability and reduce affinity to at least one Fc ligand.

FIG. 6A. Immunohistochemical staining profile of human cerebrum tissue treated with various anti-IFNAR1 antibodies. The 9D4 antibody exhibits a lower staining profile when incubated with human cerebrum tissue compared to 4G5 and MDX-1333 antibodies.

FIG. 6B. Immunohistochemical staining profile of human monocytes treated with various anti-IFNAR1 antibodies. As a positive control, various anti-IFNAR1 antibodies were tested for reactivity to human monocytes.

FIG. 7. The anti-IFNAR1 antibody 9D4 inhibits IFNα signaling in a cell based STAT activation assay. Treatment with antibody 9D4 inhibits STAT1/3/4 tyrosine phosphorylation in response to stimulation with interferon alpha as determined by Western Blot analysis with commercially available STAT antibodies.

FIG. 8. Anti-IFNAR1 antibodies block signaling of various concentrations of pDC Cell derived Type I IFNs. Presented are the IC50 values for antibody 9D4 blocking IFN signaling in a luciferase reporter assay utilizing type I IFN supernatants purified from 3 independent donors. Included are the relative amounts of IFNα, IFNβ, and IFNω in each purified type I interferon supernatant.

FIGS. 9 A, B, C. Anti-IFNAR1 antibodies 9D4, 9D4-DM (Double Mutant), and 9D4-TM (Triple Mutant) exhibit similar binding characteristics. Presented are data representing the unmodified 9D4 antibody along with 2 modified antibodies, 9D4-DM and 9D4-TM. The modified antibodies exhibit similar IFNAR1 binding characteristics to the unmodified antibody.

FIG. 10A. The anti-IFNAR1 antibody 9D4 binds soluble interferon alpha receptor (sIFNαR1). Presented are equilibrium binding data that demonstrate dose dependent binding of 9D4 to soluble interferon alpha receptor.

FIG. 10B. Determination of the Kd of 9D4 on human PBMCs. Presented is the dissociation constant determination of 9D4 measured by binding to human PBMCs.

FIG. 11. Anti-IFNAR1 antibodies inhibit IFNα induced signaling in a luciferase reporter assay. Anti-IFNAR1 antibodies including unmodified and modified antibodies demonstrate similar IC50 values for blocking Leukocyte IFN signaling in a luciferase reporter assay system.

FIG. 12A. Determination of the isoelectric point of 9D4 (unmodified) and modified 9D4 antibodies. Presented is an IEF gel documenting the relative pI values for the 9D4 WT (unmodified), 9D4-DM, and 9D4-TM antibodies.

FIG. 12B. Determination of the thermal melting temperatures of 9D4 (unmodified) and modified 9D4 antibodies. Presented here is a melt cure documenting the relative melting temperatures (Tm) for the 9D4, 9D4-DM, and 9D4-TM antibodies.

FIG. 13. Prophylactic treatment with anti-IFNAR antibodies blocks Adv-IFNα induced proteinuria. Mice treated with control vector, Adv-IFNα, Adv-IFNα+isotype control pretreatment, and Adv-IFNα+anti-IFNAR pretreatment were analyzed for proteinuria over 9 weeks. Mice pretreated with anti-IFNAR did not exhibit proteinuria after IFNα challenge.

FIG. 14. Prophylactic treatment with anti-IFNAR antibodies blocks the upregulation of IFNα responsive genes (IFIT1, IFI44, CXCL11, IFI202b, CXCL19, CXCL9) in blood. Mice pre-treated with anti-IFNAR antibodies did not exhibit not upregulated selected IFNα responsive genes upon challenge with adenovirus encoded IFN alpha as compared to mice pretreated with control virus, PBS, or isotype IgG controls. Presented are the relative expression of six genes known to be responsive to IFNα in blood samples taken from mice 3 weeks post IFNα induction by infection with Adv-IFNα.

FIGS. 15 A, B. Prophylactic treatment with anti-IFNAR antibodies blocks IFNα induced autoantibody production. Mice pre-treated with anti-IFNAR antibodies did not exhibit elevated autoantibody production upon challenge with adenovirus encoded IFNα as compared to mice pretreated with control virus, PBS or isotype IgG controls. Presented are the concentrations of anti-dsDNA and anti-SSA/Ro in blood samples taken from mice 6 weeks post IFNα induction by infection with Adv-IFNα.

FIGS. 16 A, B. Prophylactic treatment with anti-IFNAR antibodies blocks the upregulation of cytokines in the kidney. Mice pretreated with anti-IFNAR antibodies did not exhibit upregulated cytokines in the kidney upon challenge with adenovirus encoded IFN5 as compared to mice pretreated with, control virus, PBS or isotype IgG controls. Presented are the measurement of IP-10, and IL-18 levels in kidney samples taken from mice 6 weeks post IFNα induction by infection with Adv-IFNα5.

FIG. 17. Prophylactic treatment with anti-IFNAR antibodies blocks IFN induced autoantibody production. Presented here are the relative titers of anti-nuclear antigen (ANA) antibodies from mouse serum. Mice pretreated with anti-IFNAR antibodies exhibited lower ANA serum titers after IFN challenge than mice pretreated with control virus, PBS, or isotype control.

FIG. 18. Antibody mediated inhibition of SLE plasma mediated Dendritic cell development. Presented are the results of 5 individual experiments in which IFN derived from SLE patients was incubated in the presence of anti-IFNAR1 antibody 9D4 and subsequently added to human monocytes. The presence of anti-IFNAR1 antibody 9D4 inhibited the ability of IFN derived from SLE patients to induce the dendritic cell markers CD38 and CD123 in differentiating monocytes.

FIG. 19. Anti-IFNAR1 antibodies suppress the expression of CD38, CD123 and CD86 in monocytes stimulated with Leukocyte Interferon. As measured by percent suppression of control stimulated expression, anti-IFNAR1 antibodies 9D4, 9D4-DM and 9D4-TM exhibited similar inhibition profiles for the expression of CD38, CD123 and CD86 in differentiating monocytes.

FIG. 20. Modified anti-IFNAR1 antibodies exhibit decreased binding to the Fc receptor FcγRI as compared to unmodified anti-IFNAR1 antibodies. Anti-IFNAR1 antibodies 9D4 (unmodified), 9D4-DM (modified) and 9D4-TM (modified) were analyzed for the ability to bind to plate bound FcγRI in an ELISA experiment. As a positive control for Fc receptor binding, an unrelated unmodified antibody was used (control antibody).

FIGS. 21, A, B, C. Modified anti-IFNAR1 antibodies exhibit decreased binding to the Fc receptor FcγRIIIA as compared to unmodified anti-IFNAR1 antibodies. Plate bound unmodified anti-IFNAR1 antibody 9D4(A) and modified anti-IFNAR1 antibodies 9D4-DM (B) and 9D4-TM(C) were analyzed for the ability to bind free FcγRIIIA in an ELISA experimental format.

FIGS. 22, A, B, C. Modified anti-IFNAR1 antibodies exhibit decreased binding to the Fc receptor FcγRIIIA. Free unmodified anti-IFNAR1 antibody 9D4(A) and modified anti-IFNAR1 antibodies 9D4-DM(B) and 9D4-TM(C) were analyzed for the ability to bind plate bound FcγRIIIA in an ELISA experimental format.

FIGS. 23 A-E. Neutralization of IFN subtypes in SLE patient serum. As measured by reporter assay, anti-IFNAR1 antibodies MDX-1333, 9D4-WT and 9D4-TM inhibited IFN mediated signaling of α10 (A), Leukocyte interferon (B), α2b (C), ω (D), and β (E).

FIG. 24. Anti-IFNAR1 antibodies neutralize type I interferon from SLE patients. By reporter assay, the anti-IFNAR1 antibody, 9D4, inhibited type I interferon mediated signaling as compared to a control, unrelated antibody.

FIGS. 25 A-D. Anti-IFNAR antibodies suppress the IFNα induced pDC population in PBMC's. Anti-IFNAR antibodies blocked the elevation of pDC cells measured by cell surface epitope expression, induced by ectopic adenoviral induced expression of interferon alpha in spleen (A), lymph nodes (B), peripheral blood (C) and bone marrow (D).

FIG. 26. Binding analysis of anti-IFNAR1 antibodies 9D4-WT, 9D4-DM, and 9D4-TM to the Fc receptor FcγRI was determined by BIACore analysis. Briefly, anti-IFNAR1 antibodies were immobilized and free FcγRI was added to measure affinity. As demonstrated by the tracing, the modified antibodies, 9D4-DM, and 9D4-TM exhibited lower affinities to the free FcγRI as compared to the unmodified 9D4-WT antibody.

FIGS. 27 A-C. Binding analysis of anti-IFNAR1 antibodies 9D4-WT, 9D4-DM, and 9D4-TM to the Fc receptor FcγR was determined by BiaCore analysis. Briefly, free anti-IFNAR1 antibodies were passed over immobilized FcγRI to measure affinity. As demonstrated by the tracing, the modified antibodies 9D4-DM (B), and 9D4-TM (C) exhibited lower affinities to the bound FcγRI as compared to the unmodified 9D4-WT (A) antibody.

FIG. 28. Anti-IFNAR antibodies inhibit IFNα responsive gene induction in the kidney. Briefly, in the accelerated lupus mouse model, treatment with anti-IFNAR antibodies blocks induction in the kidney of six genes (ICAM1, VCAM1, CXCL9, CXCL10, and IFIT1) mediated by the ectopically expression of IFNα compared to control mice as measured by a Taqman assay.

FIG. 29. Anti-IFNAR antibodies inhibit the production of anti-ds DNA antibodies in the accelerated lupus mouse model. Briefly, mice ectopically expressing IFNα and treated with anti-IFNAR antibodies did not accumulate anti-ds DNA antibodies to the same level as mice similarly infected and treated with an IgG control antibody.

FIG. 30. Anti-IFNAR antibodies are able to reduce proteinuria in a therapeutic setting of the accelerated lupus mouse model. (A) Briefly, mice ectopically expressing IFNα developed Lupus like symptoms, such as proteinuria. In a therapeutic study, anti-IFNAR antibodies were administered to mice once a threshold proteinuria score was reached. Anti-IFNAR antibodies, PBS, or control IgG were administered semi-weekly over a 5 week time course. The anti-IFNAR antibody treated group exhibited decreased severity of proteinuria during the experiment compared to PBS only or control IgG treated groups.

FIG. 31. Anti-IFNAR antibodies are able to increase survival in a therapeutic setting of the accelerated lupus mouse model. (A) Briefly, mice ectopically expressing IFNα had a reduced survival rate at about 8 weeks after developing Lupus-like symptoms such as proteinuria. In the therapeutic study, anti-IFNAR antibodies were administered to mice once a threshold proteinuria score was reached. Anti-IFNAR antibodies, PBS, or control IgG were administered semi-weekly over a 5 week time course. After the five weeks, antibody treatment was stopped and the mortality tracked for all three treatment groups. The anti-IFNAR antibody treated group exhibited a much lower rate of mortality than the PBS alone, or control IgG groups, which both exhibited complete mortality by 9 weeks.

FIG. 32. Representation of the asymmetric unit contents of the crystals of Fc-TM that comprises L234F/L235E/P331S mutations. The mutation P331 is indicated in red. One zinc ion is chelated by two spatially close Histidine residues. The carbohydrate residues attached to 297 were modeled according to their electron density.

FIG. 33. Kinetic images demonstrate 9D4-TM internalization. THP-1 cells were stained with 1 μM CFSE in a 37° C. CO₂ incubator for 10 min followed by 1 μg/ml of Alexa647-9D4-TM on ice for 1 hr. After removal of unbound the cells were incubated at 37° C. for the times indicated (0, 15, 30 and 60 minutes) and the images of cells were taken.

FIG. 34. The anti-IFNAR1 antibody, 9D4-TM does not exhibit CDC activity in an in vitro assay. Presented in this panel are the results from a CDC assay to determine the ability of the 9D4-TM antibody to elicit CDC activity. As presented, the 9D4-TM antibody did not exhibit any CDC activity as compared to the positive control antibody. CDC activity was also undetectable for an unrelated control antibody, R347. Briefly, cells expressing IFNAR1 antigen were incubated with either the positive control antibody, 9D4-TM, or R347. After a series of washes, freshly prepared human serum was added. Complement dependent cytotoxicity (CDC) was measured using a LDH release assay.

4. TERMINOLOGY

The terms “interferon alpha”, “IFNα”, “IFNa”, “IFNA” and “IFN alpha” are used interchangeably and intended to refer to IFN alpha proteins encoded by a functional gene of the interferon alpha gene locus with 75% or greater sequence identity to IFN alpha 1 (GenBank accession number NP_076918 or protein encoded by GenBank accession number NM_024013). Examples of IFN alpha subtypes include IFN alpha 1, alpha 2a, alpha 2b, alpha 4, alpha 4b alpha 5, alpha 6, alpha 7, alpha 8, alpha 10, alpha 13, alpha 14, alpha 16, alpha 17 and alpha 21. The terms “interferon alpha”, “IFNα”, and “IFN alpha” are intended to encompass recombinant forms of the various IFN alpha subtypes, as well as naturally occurring preparations that comprise IFN alpha proteins, such as leukocyte IFN and lymphoblastoid IFN.

The terms “Interferon alpha receptor-1,”, “IFNAR1” “IFNAR-1,” and “IFNAR-1 antigen” are used interchangeably, and include variants, isoforms, species homologs of human IFNAR-1, and analogs having at least one common epitope with IFNAR-1. Accordingly, human antibodies of the invention may, in certain embodiments, cross-react with IFNAR-1 from species other than human, or other proteins which are structurally related to human IFNAR-1 (e.g., human IFNAR-1 homologs). In other embodiments, the antibodies may be completely specific for human IFNAR-1 and not exhibit species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR-1 has the Genbank accession number NM 000629.

As used herein, the term “conservative sequence modifications” is intended to include amino acid modifications that do not affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several non-limiting examples of families of conservative amino acid substitutions are shown in Table 1.

TABLE 1 Families of Conservative Amino Acid Substitutions Family Amino Acids non-polar Trp, Phe, Met, Leu, Ile, Val, Ala, Pro Uncharged polar Gly, Ser, Thr, Asn, Gln, Tyr, Cys acidic/negatively charged Asp, Glu basic/positively charged Arg, Lys, His Beta-branched Thr, Val, Ile residues that influence chain Gly, Pro orientation Aromatic Trp, Tyr, Phc, His

5. DETAILED DESCRIPTION

In contrast to previous teachings, the inventors have found that anti-IFNAR1 antibodies with reduced or ablated effector function are desired for the treatment of chronic autoimmune and/or inflammatory diseases. Previously, antibodies directed against IFNAR1 were developed with the understanding that effector function would play a role in mediating treatment or at least moderation of a chronic autoimmune and/or inflammatory disease state (see, for example U.S. Publication No. 20060029601 or PCT publication No. WO06002177). With this concept, many of the previous teachings directed the artisan to identify anti-IFNAR1 antibodies with strong effector function and to further enhance the effector function by increasing the affinity of the antibody for Fc receptors (e.g., FcRn, FcγRIIIa, FcγRIIb) and/or the complement protein C1q. These resultant effector function-enhanced anti-IFNAR1 antibodies were thought to be advantageous in the treatment of disease states.

In contrast to this previous understanding, the present invention describes anti-IFNAR1 antibodies with reduced or ablated effector function (such as ADCC and/or CDC). Through tissue cross-reactivitiy studies, it was surprisingly found that anti-IFNAR1 antibodies with strong or enhanced effector function displayed a propensity for unwanted toxicity due to the prevalence of staining of anti-IFNAR1 on non-target tissues. This toxicity would result from the non-specific activation of ADCC and/or CDC at inappropriate sites. To reduce of or eliminate this unwanted toxicity, the invetors recognized the need to reduce effector function of polypeptides comprising an Fc region.

Accordingly, one aspect of the invention encompasses modified antibodies or other polypeptides comprising the Fc region of an antibody, comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region resulting in reduced or ablated affinity for at least one Fc ligand (referred to herein as “modified antibodies of the invention”, “modified antibodies” or “antibodies of the invention”). The Fc region interacts with a number of ligands including but not limited to Fc Receptors (e.g., FcRn, FcγRIIIa, FcγRIIb), the complement protein C1q, and other molecules such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events including, but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Accordingly, in certain embodiments the modified antibodies of the invention have reduced or ablated affinity for an Fc ligand responsible for facilitating effector function compared to an antibody having the same amino acid sequence as the antibody of the invention but not comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region (also referred to herein as an “unmodified antibody”). In certain embodiments, antibodies of the invention comprise at least one or more of the following properties: reduced or ablated effector (ADCC and/or CDC) function, reduced or ablated binding to Fc receptors, or reduced or ablated toxicities. More specifically, embodiments of the invention provide anti-IFNAR1 antibodies with reduced affinity for Fc receptors (e.g., FcRn, FcγRIIIa, FcγRIIb) and/or the complement protein C1q.

In one embodiment, antibodies of the invention comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue selected from the positions consisting of: 234, 235, and 331, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). In a specific embodiment, antibodies of the invention comprise an Fc region comprising at least one amino acid substitution selected from the group consisting of: L234F, L235E, and P331 S, wherein the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position.

In another embodiment, antibodies of the invention further comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue that is correlated with increased stability of the antibody. In one embodiment, the addition, substitution, or deletion of an amino acid residue is at position 228 of the Fc region, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. In a specific embodiment, antibodies of the invention comprise an Fc region comprising an amino acid substitution at position 228, wherein the substitution is a serine residue. In another specific embodiment, antibodies of the invention of the IgG4 subtype comprise an amino acid substitution of serine at position 228 of the Fc region. In other embodiments, antibodies of the invention already comprise a serine residue at position 228 of the Fc region; in such embodiments, no modification is required. In alternative embodiments, antibodies of the invention do not require modification of residue 228 of the Fc region or already comprise serine at said position.

In another embodiment, antibodies of the invention may be any of any class (for example, but not limited to IgG, IgM, and IgE). In certain embodiments, antibodies of the invention are members of the IgG class of antibodies. In a specific embodiment, antibodies of the invention are of the IgG1 subclass. In another specific embodiment, antibodies of the invention are of the IgG1 subclass and comprise the following amino acid substitutions: 234F, 235E and 331S of the Fc region. In alternate embodiments, antibodies of the invention are of the IgG4 subclass. In a specific embodiment, antibodies of the invention are of the IgG4 subclass and comprise the following amino acid substitutions: S228P and L235E of the Fc region.

In certain embodiments, the modified antibodies of the present invention may be produced by combining a variable domain, or fragment thereof, with an Fc domain comprising one or more of the amino acid substitutions disclosed herein. In other embodiments modified antibodies of the invention may be produced by modifying an Fc domain-containing antibody by introducing one or more of the amino acid substitutions residues into the Fc domain.

5.1 Reduced Binding to Fc Ligands

One skilled in the art will understand that antibodies of the invention may have altered (relative to an unmodified antibody) FcγR and/or C1q binding properties (examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K_(D)), dissociation and association rates (K_(off) and K_(on) respectively), binding affinity and/or avidity) and that certain alterations are more or less desirable. It is known in the art that the equilibrium dissociation constant (K_(D)) is defined as k_(off)/k_(on). One skilled in the art can determine which kinetic parameter is most important for a given antibody application. For example, a modification that reduces binding to one or more positive regulator (e.g., FcγRIIIA) and/or enhanced binding to an inhibitory Fc receptor (e.g., FcγRIIB) would be suitable for reducing ADCC activity. Accordingly, the ratio of binding affinities (e.g., equilibrium dissociation constants (K_(D))) can indicate if the ADCC activity of an antibody of the invention is enhanced or decreased. Additionally, a modification that reduces binding to C1q would be suitable for reducing or eliminating CDC activity.

The affinities and binding properties of an Fc region for its ligand, may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-FcγR interactions, i.e., specific binding of an Fc region to an FcγR including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4^(th) Ed., Lippincott-Raven, Philadelphia (1999).

In one embodiment, antibodies of the invention exhibit reduced binding affinity for one or more Fc receptors including, but not limited to FcγRI (CD64) including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32 including isoforms FcγRIIA, FcγRIIB, and FcγRIIC); and FcγRIII (CD16, including isoforms FcγRIIIA and FcγRIIB) as compared to an unmodified antibody. In certain embodiments, antibodies of the invention do not comprise a concomitant increase in binding the FcγRIIB receptor as compared to an unmodified (for example, containing a wild type Fc region) antibody.

In one embodiment, antibodies of the invention exhibit decreased affinities to FcγRI relative to an unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for FcγRI receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than an unmodified antibody.

In another embodiment, antibodies of the invention exhibit affinity for FcγRI receptor that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.

In one embodiment, antibodies of the invention exhibit decreased affinity for the FcγRIIIA receptor relative to an unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for FcγRIIIA receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than an unmodified antibody.

In another embodiment, antibodies of the invention exhibit affinities for FcγRIIIA receptor that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.

It is understood in the art that the F158V allelic variant of the FcγRIIIA receptor has altered binding characteristics to antibodies. In one embodiment, antibodies of the invention bind with decreased affinities to FcγRIIIA (F158V) relative to an unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for FcγRIIIA (F158V) receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of an unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for the FcγRIIIA(F158V) receptor that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.

In another embodiment, antibodies of the invention exhibit increased affinities for the FcγRIIB receptor as compared to unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for the FcγRIIB receptor that are unchanged or increased by at least at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold than that of an unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for the FcγRIIB receptor that are increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% than an unmodified antibody.

In another embodiment, antibodies of the invention exhibit affinities for the FcγRI, FcγRIIIA, or FcγRIIIA (F158V) receptors that are between about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In certain embodiments, antibodies of the invention exhibit affinities for the FcγRI, FcγRIIIA, or FcγRIIIA (F158V) receptors that are greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM.

In another embodiment, antibodies of the invention exhibit affinities for the FcγRIIB receptor that are between about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In certain embodiments, antibodies of the invention exhibit affinities for the FcγRI, FcγRIIIA, or FcγRIIIA (F158V) receptors that are less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 2.5 μM, less than 1 μM, or less than 100 nM, or less than 10 nM.

In another embodiment, antibodies of the invention exhibit affinities for the FcγRIIB receptor that are between about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In certain embodiments, antibodies of the invention exhibit affinities for the FcγRI, FcγRIIIA, or FcγRIIIA (F158V) receptors that are less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 2.5 μM, less than 1 μM, or less than 100 nM, or less than 10 nM.

5.2 Reduced ADCC Activity

It is well known in the art that antibodies are capable of directing the attack and destruction of targeted antigen through multiple processes collectively known in the art as antibody effector functions. One of these processes, known as “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of target cells “arm” the cytotoxic cells and are required for such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement. Another process encompassed by the term effector function is complement dependent cytotoxicity (hereinafter referred to as “CDC”) which refers to a biochemical event of antibody-mediated target cell destruction by the complement system. The complement system is a complex system of proteins found in normal blood plasma that combines with antibodies to destroy pathogenic bacteria and other foreign cells.

The ability of any particular antibody to mediate lysis of the target cell by ADCC can be assayed. To assess ADCC activity an antibody of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361; Wilkinson et al., 2001, J Immunol Methods 258:183-191; Patel et al., 1995 J Immunol Methods 184:29-38. Alternatively, or additionally, ADCC activity of the antibody of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA 95:652-656.

It is contemplated that antibodies of the invention are characterized by in vitro functional assays for determining one or more FcγR mediated effector cell functions. In certain embodiments, antibodies of the invention have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude antibodies of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.

In one embodiment, antibodies of the invention exhibit decreased ADCC activities as compared to an unmodified antibody. In another embodiment, antibodies of the invention exhibit ADCC activities that are at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold less than that of an unmodified antibody. In still another embodiment, antibodies of the invention exhibit ADCC activities that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody. In certain embodiments, antibodies of the invention have no detectable ADCC activity. In specific embodiments, the reduction and/or ablatement of ADCC activity may be attributed to the reduced affinity antibodies of the invention exhibit for Fc ligands and/or receptors.

5.3 Reduced CDC Activity

The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule, an antibody for example, complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163, may be performed.

In one embodiment, antibodies of the invention exhibit decreased affinities to C1q relative to an unmodified antibody. In another embodiment, antibodies of the invention exhibit affinities for C1q receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than an unmodified antibody.

In another embodiment, antibodies of the invention exhibit affinities for C1q that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.

In another embodiment, antibodies of the invention exhibit affinities for C1q that are between about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In certain embodiments, antibodies of the invention exhibit affinities for C1q that are greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM.

In one embodiment, antibodies of the invention exhibit decreased CDC activities as compared to an unmodified antibody. In another embodiment, antibodies of the invention exhibit CDC activities that are at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold less than that of an unmodified antibody. In still another embodiment, antibodies of the invention exhibit CDC activities that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody. In certain embodiments, antibodies of the invention exhibit no detectable CDC activities. In specific embodiments, the reduction and/or ablatement of CDC activity may be attributed to the reduced affinity antibodies of the invention exhibit for Fc ligands and/or receptors.

5.4 Reduced Antibody Related Toxicity

It is understood in the art that biological therapies may have adverse toxicity issues associated with the complex nature of directing the immune system to recognize and attack unwanted cells and/or targets. When the recognition and/or the targeting for attack do not take place where the treatment is required, consequences such as adverse toxicity may occur. For example, antibody staining of non-targeted tissues may be indicative of potential toxicity issues.

In one embodiment, antibodies of the invention exhibit reduced staining of non-targeted tissues as compared to an unmodified antibody. In another embodiment, antibodies of the invention exhibit reduced staining of non-targeted tissues that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of an unmodified antibody. In another embodiment, antibodies of the invention exhibit reduced staining of non-targeted tissues that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody.

In one embodiment, antibodies of the invention exhibit a reduced antibody related toxicity as compared to an unmodified antibody. In another embodiment, antibodies of the invention exhibit toxicities that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of an unmodified antibody. In another embodiment, antibodies of the invention exhibit toxicities that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody.

5.5 Internalizing Antibodies

Antibodies of the invention may bind to cell-surface antigens that may internalize, further carrying the antibodies into the cell. Once inside the cell, the antibodies may be released into the cytoplasm, targeted to a specific compartment, or recycled to the cell surface. In some embodiments, the antibodies of the invention bind to a cell-surface antigen that internalizes. In other embodiments, antibodies of the invention may be targeted to specific organelles or compartments of the cell. In yet other embodiments, the antibodies of the invention may be recycled to the cell surface or periphery after internalization. In a specific embodiment, the antibody of the invention is specific for IFNAR1.

Internalization of antibodies may be measured by art-accepted techniques such as those presented in Example 34. In some embodiments, the extent of internalization is represented as a percentage of total antibody bound to cells. In other embodiments, the extent of antibody internalization is represented as a comparison to a non-specific control antibody. In other embodiments, the extent of antibody internalization is represented as a comparison to an antibody that binds a cell-surface antigen that does not internalize. In yet other embodiments, the extent of antibody internalization is correlated with the degradation of the antibody. In yet other embodiments, the extent of antibody internalization is represented as a ratio of cytoplasmic versus cell surface staining.

In one embodiment, the antibodies of the invention once bound, internalize into cells wherein internalization is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, or at least about 170% more than a non-specific control antibody.

In another embodiment, the antibodies of the invention once bound, internalize into cells wherein internalization is 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170% more than a non-specific control antibody.

In another embodiment, the antibodies of the invention once bound, internalize into cells wherein internalization is 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170% more than control antibodies as determined by the internalization assay using a secondary antibody.

5.6 Three-Dimensional Structure of a Human Fc Region

The present invention also provide crystalline forms of a human IgG Fc region, wherein the human Fc region, designated as Fc-TM, comprises amino acid substitutions of L234F, L235E and P331S as numbered by the EU index as set forth in Kabat and exhibits reduced or ablated effector (ADCC and/or CDC) function, reduced or ablated binding to Fc receptors, and/or reduced or ablated toxicities. In certain embodiments, the crystals are characterized by an orthorhombic space group C222₁ with unit cell of a=50.18, b=147.30 and c=75.47. In certain embodiments, the crystals are of diffraction quality to permit the determination of the three-dimensional X-ray diffraction structure of the crystalline polypeptide(s) to high resolution, preferably to a resolution of greater than about 3 Å, typically in the range of about 2 Å to about 3 Å.

The present invention further provides the high-resolution three-dimensional structures and atomic structure coordinates of the Fc-TM crystals. The specific methods used to obtain crystals and structure coordinates are provided in the examples, infra.

The atomic structure coordinates of crystalline Fc-TM, obtained from the C222₁ form of the crystal to 2.3 Å resolution, are listed in Table 6. All residues at positions 236 to 445 could be traced in the electron density and no electron density was observed for hinge residues prior to position 236, including the L234F and L235E mutations. The electron density at position 331 corresponded to serine.

The overall three-dimensional structure of Fc-TM was very similar to previously reported structures of unliganded human Fc regions (Deisenhofer, (1981). Biochemistry, 20, 2361-2370; Krapp et al., (2003). J. Mol. Biol. 325, 979-989; Matsumiya et al., (2007). J. Mol. Biol. 368, 767-779; Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, in press). When considered individually, Fc-TM C_(H)2 and C_(H)3 domains showed great structural conservation and rigidity when compared with other unliganded, unmutated human Fc structures.

The structure information can be used in a variety of computation or computer-based methods to screen, design or identify anti-IFNAR antibodies that have alter biological properties. For example, the crystals and structure coordinates obtained therefrom can be used to screen, design or identify amino acid additions, substitutions or deletions in Fc region that result in reduced or ablated binding to Fc receptors, reduced or ablated effector (ADCC and/or CDC) function, or reduced or ablated toxicitics.

Once an antibody has been designed or selected by the above methods, its effector function, binding to Fc receptors, or toxicities may be tested and optimized by any methods known to those of skill in the art. Exemplary methods are described in sections 5.1-5.4 above.

The present invention also encompasses anti-IFNAR1 antibodies that are designed or selected by the use of the structure information of Fc-TM and that exhibit the desired biological activities. In some embodiments, such antibodies comprise an Fc region with the mutations of L234F, L235E, and P331S. In some embodiments, such antibodies comprise an Fc region with one or more addition, substitution, or deletion of an amino acid residue other than amino acid residues 234, 235, and 331.

5.7 Anti-IFNAR1 Antibodies

In one embodiment, antibodies of the invention are specific for (i.e. specifically bind) IFNAR1. Such antibodies may also be referred to herein as “anti-IFNAR1 antibodies of the invention.” In another embodiment, antibodies of the invention are specific for human IFNAR1. In another embodiment, the anti-IFNAR1 antibodies of the invention may cross-react with IFNAR1 from species other than human, or other proteins which are structurally related to human IFNAR1 (for example, human IFNAR1 homologs). In other embodiments, the anti-IFNAR1 antibodies of the invention may be specific for human IFNAR1 only and not exhibit species or other types of cross-reactivity.

In one embodiment, the anti-IFNAR1 antibodies of the invention exhibit reduced binding affinities for Fc ligands and have at least one of the following properties: reduced or ablated effector (ADCC and/or CDC) function, reduced or ablated binding to Fc ligands, or reduced or ablated toxicities as compared to an unmodified antibody.

In one embodiment, anti-IFNAR1 antibodies of the invention comprise the addition, substitution or deletion of at least one amino acid residue selected from the group consisting of: L234F, L235E, and P331 S. In a specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the amino acid substitutions: L234F, L235E, and P331S of the Fc region. In a specific embodiment, an anti-IFNAR1 antibody of the invention is an IgG isotype antibody.

In another embodiment, anti-IFNAR1 antibodies of the invention are of the IgG4 subclass. In yet another embodiment, anti-IFNAR1 IgG4 antibodies of the invention comprise the amino acid substitution L235E of the Fc region. In another embodiment, the anti-IFNAR1 IgG4 antibodies of the invention also comprise an amino acid change that is correlated with increased stability. In a specific embodiment, anti-IFNAR1 IgG4 antibodies of the invention further comprise the amino acid substitution S228P of the Fc region.

In another embodiment, anti-IFNAR1 antibodies of the invention exhibit reduced or ablated binding affinities for Fc receptors (for example, but not limited to FcγRI (CD64), including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32), including isoforms FcγRIIA, FcγRIIB, and FcγRIIC; and FcγRIII (CD16), including isoforms FcγRIIIA and FcγRIIB) as compared to an unmodified antibody. In a specific embodiment, the anti-IFNAR 1 antibodies of the invention exhibit decreased affinities to FcγRI relative to an unmodified antibody. In another specific embodiment, the anti-IFNAR1 antibodies of the invention exhibit decreased affinities for the FcγRIIIA receptor relative to an unmodified antibody. In another specific embodiment, the anti-IFNAR1 antibodies of the invention bind with decreased affinities to the F158V allele of FcγRIIIA relative to an unmodified antibody.

In another embodiment, anti-IFNAR1 antibodies of the invention exhibit reduced or ablated binding affinities for C1q as compared to an unmodified antibody. In a specific embodiment, the anti-IFNAR 1 antibodies of the invention exhibit decreased affinities to FcγRI relative to an unmodified antibody.

In one embodiment, anti-IFNAR1 antibodies of the invention exhibit reduced or ablated effector function. In a specific embodiment, anti-IFNAR1 antibodies of the invention exhibit reduced or ablated ADCC and/or CDC activity. In another specific embodiment, the anti-IFNAR1 antibodies of the invention exhibit reduced or ablated toxicity.

5.7.1 Anti-IFNAR1 Antibody Sequences

In one embodiment, the amino acid sequences of the heavy chain variable regions and/or light chain variable regions of the anti-IFNAR1 antibodies of the invention are provided herein as FIGS. 1A, 2A, 3A, 4A and FIGS. 1B, 2B, 3B, 4B respectively. In another embodiment, the polynucleotide sequence encoding the heavy chain variable and light chain variable regions of the anti-IFNAR1 antibodies of the invention are provided herein as FIGS. 1A, 2A, 3A, 4A and FIGS. 1B, 2B, 3B, 4B respectively.

In another embodiment, selected sequences of anti-IFNAR1 antibodies of the invention can be found in U.S. Pat. No. 5,919,453, U.S. patent application Ser. Nos. 10/831,459, 10/182,058, 11/157,494, and 11/521,102 each of which are incorporated by reference in their entireties for all purposes. In an alternative embodiment, the sequences of the anti-IFNAR1 antibodies of the invention do not comprise the sequences found in U.S. Pat. No. 5,919,453, U.S. patent application Ser. Nos. 10/831,459, 10/182,058, 11/157,494, and 11/521,102.

In other embodiments, antibodies of the invention are disclosed in U.S. Patent Provisional Application Ser. Nos. 60/842,925, filed Sep. 8, 2006, 60/866,917; filed Nov. 22, 2006; 60/911,397, filed Apr. 12, 2007; 60/915,309, filed May 22, 2007; U.S. patent application Ser. No. 11/852,106, filed Sep. 7, 2007; and PCT Application Serial No. US2007/07791, filed Sep. 7, 2007, each of which are incorporated in its entirety for all purposes.

In one embodiment, anti-IFNAR1 antibodies of the invention also include antibodies that comprise an amino acid sequence of a variable heavy chain and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the variable heavy chain and/or light chain of the 3F11, 11E2, 4G5, and 9D4 antibodies (see FIGS. 1-4 for sequences).

It will be understood that the complementarity determining regions (CDRs) residue numbers referred to herein are those of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). Specifically, residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of “spacer” residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al. supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.

In one embodiment, the anti-IFNAR1 antibodies of the invention comprise at least one VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 2. In another embodiment, the anti-IFNAR1 antibodies of the invention comprise at least one VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 2. In other embodiments, the anti-IFNAR1 antibodies of the invention comprise one or more of the VH CDRs and one or more of the VL CDRs listed in Table 2. In still other embodiments, the anti-IFNAR1 antibodies of the invention comprise any combination of the VH CDRs and VL CDRs listed in Table 2. In another embodiment, the anti-IFNAR1 antibodies of the invention may comprise at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6 CDRs selected from Table 2. In another embodiment, anti-IFNAR1 antibodies of the invention may comprise a VH domain and/or a VL domain each comprising 1, 2 or 3 CDRs. In another embodiment, the anti-IFNAR1 antibodies of the invention may comprise a VH further comprising 1, 2, or 3 heavy chain CDRs (CDRH#) listed in Table 2. In another embodiment, the anti-IFNAR1 antibodies of the invention may comprise a VL further comprising 1, 2, or 3 light chain CDRs (CDRL#) listed in Table 2.

In a specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 3F11 (see for example Table 2). In another specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 4G5 (see for example Table 2). In another specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 11E2 (see for example Table 2). In yet another specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 9D4 (see for example Table 2).

TABLE 2 Anti-IFNAR1 antibody CDR sequences Antibody CDR Sequence Seq ID No: 3F11 CDRL1 RASQGIYSVLA 1 3F11 CDRL2 DASRLES 2 3F11 CDRL3 QQFNSYIT 3 3F11 CDRH1 GYFWS 4 3F11 CDRH2 EIDHSGKTNYNPSLKS 5 3F11 CDRH3 ESKYYFGLDV 6 4G5 CDRL1 RATQDISIALV 11 4G5 CDRL2 DASGLGS 12 4G5 CDRL3 QQFNSYPYT 13 4G5 CDRH1 NYYWS 14 4G5 CDRH2 EIILSGSTNYNPSLKS 15 4G5 CDRH3 ESKWGYYFDS 16 11E2 CDRL1 RASQSVSSSFFA 21 11E2 CDRL2 GASSRAT 22 11E2 CDRL3 QQYYDSSAIT 23 11E2 CDRH1 NYWIA 24 11E2 CDRH2 IIYPGDSDIRYSPSFQG 25 11E2 CDRH3 HDIEGFDY 26 9D4 CDRL1 RASQSVSSSFFA 31 9D4 CDRL2 GASSRAT 32 9D4 CDRL3 QQYDSSAIT 33 9D4 CDRH1 NYWIA 34 9D4 CDRH2 IIYPGDSDIRYSPSFQG 35 9D4 CDRH3 HDIEGFDY 36

In one embodiment, anti-IFNAR1 antibodies of the invention comprise an amino acid sequence of a variable heavy chain and/or variable light chain that comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 amino acid substitutions, additions, or deletions as compared to the variable heavy chains and/or light chains represented in FIG. 1, 2, 3, or 4. In another embodiment, anti-IFNAR1 antibodies of the invention comprise one or more CDRs with at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid substitutions, deletions, or additions of one or more CDRs listed in Table 2.

In another embodiment, anti-IFNAR1 antibodies of the invention comprise antibodies encoded by a polynucleotide sequence that hybridizes to the nucleotide sequence represented in FIG. 1, 2, 3, or 4 under stringent conditions. In another embodiment, anti-IFNAR1 antibodies of the invention comprise one or more CDRs encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of one or more CDRs listed in FIG. 1, 2, 3, or 4. Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., highly stringent conditions such as hybridization to filter-bound DNA in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 60° C., or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F. M. et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3). In another embodiment, anti-IFNAR1 antibodies of the invention include, but are not limited to antibodies encoded by a polynucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide sequence encoding antibodies 3F11, 11E2, 4G5, or 9D4 (see FIGS. 1-4).

5.7.2 Anti-IFNAR1 Binding Affinity

In certain embodiments, the anti-IFNAR1 antibodies of the invention exhibit a high binding affinity for IFNAR1. In a specific embodiment, anti-IFNAR1 antibodies of the invention exhibit association rate (k_(on)) of at least 10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least 10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, or at least 10⁸ M⁻¹s⁻¹. In another embodiment, anti-IFNAR1 antibodies of the invention exhibit a k_(on) of at least 2×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least 10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷ M⁻¹s⁻¹, or at least 10⁸ M⁻¹s⁻¹.

In another embodiment, anti-IFNAR1 antibodies of the invention exhibit a dissociation rate (k_(off)) of less than 10⁻¹ s⁻¹, less than 5×10⁻¹ s⁻¹, less than 10⁻² s⁻¹, less than 5×10⁻² s⁻¹, less than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰⁻¹ s⁻¹. In another embodiment, anti-IFNAR1 antibodies of the invention exhibit a k_(off), of less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In another embodiment, anti-IFNAR1 antibodies of the invention exhibit an affinity constant or K_(a) (k_(on)/k_(off)) of at least 10² M⁻¹, at least 5×10² M⁻¹, at least 10³ M⁻¹, at least 5×10³ M⁻¹, at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵ M⁻¹, at least 5×10⁵ M⁻¹, at least 10⁶ M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, at least 5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹ M⁻¹, at least 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹² M⁻¹, at least 10¹³ M⁻¹, at least 5×10¹³ M⁻¹, at least 10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or at least 5×10¹⁵ M⁻¹.

In another embodiment, anti-IFNAR1 antibodies of the invention exhibit a dissociation constant or K_(d) (k_(oft)/k_(on)) of less than 10⁻² M, less than 5×10⁻² M, less than 10⁻³ M, less than 5×10⁻³ M, less than 10⁻⁴ M, less than 5×10⁻⁴ M, less than 10⁻⁵ M, less than 5×10⁻⁵ M, less than 10⁻⁶ M, less than 5×10⁶ M, less than 10⁻⁷ M, less than 5×10⁻⁷ M, less than 10⁻⁸ M, less than 5×10⁸ M, less than 10⁻⁹ M, less than 5×10⁻⁹ M, less than 10⁻¹⁰ M, less than 5×10⁻¹⁰ M, less than 10⁻¹¹ M, less than 5×10⁻¹¹ M, less than 10⁻¹² M, less than 5×10⁻¹² M, less than 10⁻¹³ M, less than 5×10⁻¹³ M, less than 10⁻¹⁴ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁵ M, or less than 5×10⁻¹⁵ M.

5.7.3 Interferon Alpha Subtype Specificity

In one embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of one ore more Type I interferon (IFN) including, but not limited to, IFNα, IFNβ, and IFNω. Binding of IFNα subtypes can be determined by routine competition assays such as that described in Antibodies: A Laboratory Manual, CSHL. In another embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of, including but not limited to, IFNα, IFNβ, and IFNω. In another embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of one or more subtypes of IFN including, but not limited to, IFNα subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21. In another embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of all subtypes of IFNα. In this context, anti-IFNAR1 antibodies of the invention exhibit the ability to block the binding of and/or neutralize the biological activity of IFNα subtypes IFNα 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21. In one embodiment, anti-IFNAR1 antibodies of the invention do not exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of one or more subtypes of IFNα including, but not limited to, IFNα subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21. In a specific embodiment, anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity all IFNα subtypes except IFNα21.

In another embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of the following IFNα subtypes: 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21. In an alternative embodiment, the anti-IFNAR1 antibodies of the invention do not exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of the following IFNα subtypes: 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.

In other embodiments, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of non-naturally occurring type I-like interferons. Such non-naturally occurring type I-like interferons, or hybrid type I-like interferons represent molecules that have been altered from their naturally occurring structures by recombinant or synthetic techniques. Hybrid interferons, as described in U.S. Pat. No. 7,232,563, represent a molecular replacement of various segments of a naturally occurring interferon structure to create a molecule that has increased potency and/or reduced toxicity.

In other embodiments, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of mutated type I interferons. Mutated type I interferons are described in U.S. Pat. Nos. 6,299,870 and 6,300,474 which are incorporated by reference in their entireties.

In yet other embodiments, the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of type I-like interferons derived from other animal species. Such type I-like interferons are isolated from chicken, cat, mouse, rat, rabbit, goat, horse or other animal species. In specific embodiments, human type I interferons are isolated from cells derived from chicken, cat, mouse, rat, rabbit, goat, horse or other animal species. In other embodiments, human type I interferons entail different glycosylation patterns when derived from chicken, cat, mouse, rat, rabbit, goat, horse or other animal species. Further discussion of interferons from other animal species can be found in WIPO publication No. WO06099451A3 which is hereby incorporated by reference.

For the purpose of the present invention, the ability of anti-IFNAR1 antibodies of the invention to neutralize the activity of IFNα, can be monitored, for example, in a Kinase Receptor Activation (KIRA) Assay as described in WO 95/14930, published Jun. 1, 1995, by measuring the ability of a candidate antibody to reduce tyrosine phosphorylation (resulting from ligand binding) of the IFNAR1/R2 receptor complex.

Alternatively, or optionally, for the purpose of the present invention, the ability of anti-IFNAR1 antibodies of the invention to neutralize the elicitation of a cellular response by IFNα may be tested by monitoring the neutralization of the antiviral activity of IFNα, as described by Kawade, J. Interferon Res. 1:61 70 (1980), or Kawade and Watanabe, J. Interferon Res. 4:571 584 (1984), or Yousefi, et al., Am. J. Clin. Pathol. 83: 735 740 (1985), or by testing the ability of anti-IFNAR1 antibodies of the invention to neutralize the ability of IFN to activate the binding of the signaling molecule, interferon-stimulated factor 3 (ISGF3), to an oligonucleotide derived from the interferon-stimulated response element (ISRE), in an electrophoretic mobility shift assay, as described by Kurabayashi et al., Mol. Cell Biol., 15: 6386 (1995).

In one embodiment, anti-IFNAR1 antibodies of the invention exhibit the ability to inhibit at least one IFNα mediated function of the IFNAR1 receptor. In one embodiment, the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFNα or subtypes thereof by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment, the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFNα or subtypes thereof as measured by the KIRA assay described above by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment, the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFNα or subtypes thereof as measured by the binding of the signaling molecule, interferon-stimulated factor 3 (ISGF3), to an oligonucleotide derived from the interferon-stimulated response element (ISRE), in an electrophoretic mobility shift assay, as described by Kurabayashi et al., Mol. Cell Biol., 15: 6386 (1995) by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment, the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFNα or subtypes thereof as measured by an assay known in the art by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

In another embodiment, anti-IFNAR1 antibodies of the invention exhibit the ability to neutralize the anti-viral properties of IFNα or subtypes thereof. In one embodiment, the anti-IFNAR1 antibodies of the invention neutralize at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the anti-viral activity of IFN or subtypes thereof, as determined by the anti-viral assay of Kawade (1980), or Yousefi (1985). In an alternative embodiment, anti-IFNAR1 antibodies of the invention do not neutralize the anti-viral properties of IFNα or subtypes thereof.

For the purpose of the present invention, the ability of anti-IFNAR1 antibodies of the invention to block the binding of IFNα or subtypes thereof to IFNAR1 can be determined by a routine competition assay such as that described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). In one embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block or inhibit binding of the following IFNα subtypes: 1, 2, 4, 5, 8, 10, and 21 to IFNAR1. In another embodiment, the anti-IFNAR1 antibodies of the invention exhibit the ability to block ore inhibit binding of: at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of the following IFNα subtypes: 1, 2, 4, 5, 8, 10, and 21 to IFNAR1.

Antibodies of the invention may act on IFNAR to regulate IFN-I responsive genes. IFN-I responsive genes have been identified in US Patent Applications entitled “IFN alpha-induced Pharmacodynamic Markers” with the following serial numbers; 60/873,008, filed Dec. 6, 2006; 60/907,762, filed Apr. 16, 2007; 60/924,584, filed May 21, 2007; 60/960,187, filed Sep. 19, 2007; 60/966,176, filed Nov. 5, 2007 and PCT application serial number PCT/US2007/02494, filed Dec. 6, 2007 each of which are incorporated by reference in their entireties.

5.7.4 Antibodies

Antibodies of the invention may include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. As outlined herein, the terms “antibody” and “antibodies” specifically include the modified antibodies described herein. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Antibodies of the invention can be of any isotype. In one embodiment, antibodies of the invention are of the IgG1, IgG2, IgG3 or IgG4 isotype. Antibodies of the invention can be full-length antibodies comprising variable and constant regions, or they can be antigen-binding fragments thereof, such as a single chain antibody.

The term “antigen-binding fragment” of an antibody (or simply “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IFNAR1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V_(L) and V_(H), are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_(L) and V_(H) regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

In other embodiments, the invention provides fusion proteins (hereinafter referred to as “fusion proteins of the invention”) comprising a modified Fc region with reduced or ablated affinity for an Fc ligand responsible for facilitating effector function compared to an Fc region having the same amino acid sequence as the fusion protein of the invention but not comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region.

In some embodiments, fusion proteins of the invention may comprise a peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or an antibody mimetic fused to a modified Fc region. In some embodiments, fusion proteins of the invention comprise a linker region connecting the peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or an antibody mimetic to the modified Fc region. The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995), Protein Eng. 8, 725-731; Robinson & Sauer (1996), Biochemistry 35, 109-116; Khandekar et al. (1997), J. Biol. Chem. 272, 32190-32197; Fares et al. (1998), Endocrinology 139, 2459-2464; Smallshaw et al. (1999), Protein Eng. 12, 623-630; U.S. Pat. No. 5,856,456).

In one embodiment, fusion proteins of the invention comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue selected from the group consisting of: 234, 235, and 331, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). In a specific embodiment, fusion proteins of the invention comprise an Fc region comprising at least one amino acid residue selected from the group consisting of: L234F, L235E, and P331S.

In another embodiment, fusion proteins of the invention further comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue that is correlated with increased stability of the fusion protein. In one embodiment, the addition, substitution, or deletion of an amino acid residue is at position 228 of the Fc region, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (supra). In a specific embodiment, fusion proteins of the invention comprise an Fc region comprising an amino acid substitution at position 228, wherein the substitution is a serine residue.

In some embodiments, the antibodies or fusion proteins of the present invention comprise one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to a molecule comprising an Fc region. Engineered glycoforms may be useful for a variety of purposes, including but not limited to reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49 each of which is incorporated herein by reference in its entirety.

5.7.5 Antibody Conjugates

The present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.

The present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452, which are incorporated by reference in their entireties.

Additional fusion proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be modified by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to IFNAR1 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification. In other embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the “Flag” tag.

In other embodiments, antibodies of the present invention or fragments, analogs or derivatives thereof conjugated to a diagnostic or detectable agent. Such antibodies can be useful for monitoring or prognosing the development or progression of an inflammatory disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹¹⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin; positron emitting metals using various positron emission tomographies, non-radioactive paramagnetic metal ions, and molecules that are radiolabelled or conjugated to specific radioisotopes.

Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62:119-58.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated to an antibody or fragment thereof that specifically binds to IFNAR1 should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject. A clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to an antibody or fragment thereof that specifically binds to IFNAR1: the nature of the disease, the severity of the disease, and the condition of the subject.

5.7.6 Methods of Producing Antibodies

The antibodies or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and known in the art. Briefly, mice can be immunized with IFNAR1 and once an immune response is detected, e.g., antibodies specific for IFNAR1 are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, monoclonal antibodies can be generated by culturing a hybridoma cell secreting an antibody of the invention wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with IFNAR1 with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind IFNAR1.

Antibody fragments which recognize specific IFNAR1 epitopes may be generated by any technique known to those of skill in the art. For example, Fab and F(ab′)₂ fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. Further, the antibodies of the present invention can also be generated using various phage display methods known in the art.

In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to the IFNAR1 epitope of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; International Application No. PCT/GB91/01134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in International Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043 (said references incorporated by reference in their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g. the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. In certain embodiments, the vectors for expressing the VH or VL domains comprise an EF-1 alpha promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be advantageous to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,311,415, which are incorporated herein by reference in their entirety.

A humanized antibody is an antibody or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)₂, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In certain instances, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Ordinarily, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD; IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Usually the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit: cytotoxic activity and the class is typically IgG₁. Where such cytotoxic activity is not desirable, the constant domain may be of the IgG₂ class. The humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art. The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and possibly greater than 95%. Humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119-25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267-79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895-904 (1996), Couto et al., Cancer Res. 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu J S, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter or improve antigen binding. These framework substitutions are identified by methods known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323, which are incorporated herein by reference in their entireties.)

5.7.7 Polynucleotides Encoding an Antibody

The invention also encompass polynucleotides that hybridize under high stringency, intermediate or lower stringency hybridization conditions, e.g., as defined above, to polynucleotides that encode an antibody of the invention.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Since the amino acid sequences of the antibodies are known, nucleotide sequences encoding these antibodies can be determined using methods known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody or fragment thereof of the invention. Such a polynucleotide encoding the antibody maybe assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmejer et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, usually poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method known in the art.

Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, one or more of the CDRs is inserted within framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and in certain instances human framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a listing of human framework regions). Optionally, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to IFNAR1. Optionally, one or more amino acid substitutions may be made within the framework regions, and, in certain instances, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In specific embodiments, antibodies of the invention are encoded by polynucleotide sequences exemplified in FIGS. 1-4. In other specific embodiments, polynucleotides of the invention encode antibodies comprising light chain and heavy chain constant regions corresponding to SEQ ID Nos: 41 and 42 respectively. In yet other specific embodiments, polynucleotides of the invention encode antibodies comprising heavy chain constant regions corresponding to SEQ ID No: 42 with an allowance for allelic variation wherein the variation is at least one or more residue selected from the group consisting of positions 214, 221, 356, and 358 as defined by the EU index numbering system.

5.7.8 Recombinant Expression of an Antibody

Recombinant expression of an antibody of the invention, derivative, analog or fragment thereof, (e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (but not necessarily containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication No. WO 86/05807; International Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody maybe cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In other embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In certain embodiments bacterial cells such as Escherichia coli, and in other embodiments, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2). In a specific embodiment, the expression of nucleotide sequences encoding antibodies or fragments thereof which specifically bind to IFNAR1 is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH 11(5):155-2 15); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N Y (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, N Y (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences for the heavy and light chains may comprise eDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

5.8 Scalable Production of Antibodies

In an effort to obtain large quantities, antibodies of the invention may be produced by a scalable process (hereinafter referred to as “scalable process of the invention”). In some embodiments, antibodies may be produced by a scalable process of the invention in the research laboratory that may be scaled up to produce the antibodies of the invention in analytical scale bioreactors (for example, but not limited to 5 L, 10 L, 15 L, 30 L, or 50 L bioreactors). In other embodiments, the antibodies may be produced by a scalable process of the invention in the research laboratory that may be scaled up to produce the antibodies of the invention in production scale bioreactors (for example, but not limited to 75 L, 100 L, 150 L, 300 L, or 500 L). In some embodiments, the scalable process of the invention results in little or no reduction in production efficiency as compared to the production process performed in the research laboratory. In other embodiments, the scalable process of the invention produces antibodies at production efficiency of about 10 mg/L, about 20 m/L, about 30 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 250 mg/L, about 300 mg/L or higher. In other embodiments, fusion proteins may be produced by scalable processes of the invention.

In other embodiments, the scalable process of the invention produces antibodies at production efficiency of at least about 10 mg/L, at least about 20 m/L, at least about 30 mg/L, at least about 50 mg/L, at least about 75 mg/L, at least about 100 mg/L, at least about 125 mg/L, at least about 150 mg/L, at least about 175 mg/L, at least about 200 mg/L, at least about 250 mg/L, at least about 300 mg/L or higher.

In other embodiments, the scalable process of the invention produces antibodies at production efficiency from about 10 mg/L to about 300 mg/L, from about 10 mg/L to about 250 mg/L, from about 10 mg/L to about 200 mg/L, from about 10 mg/L to about 175 mg/L, from about 10 mg/L to about 150 mg/L, from about 10 mg/L to about 100 mg/L, from about 20 mg/L to about 300 mg/L, from about 20 mg/L to about 250 mg/L, from about 20 mg/L to about 200 mg/L, from 20 mg/L to about 175 mg/L, from about 20 mg/L to about 150 mg/L, from about 20 mg/L to about 125 mg/L, from about 20 mg/L to about 100 mg/L, from about 30 mg/L to about 300 mg/L, from about 30 mg/L to about 250 mg/L, from about 30 mg/L to about 200 mg/L, from about 30 mg/L to about 175 mg/L, from about 30 mg/L to about 150 mg/L, from about 30 mg/L to about 125 mg/L, from about 30 mg/L to about 100 mg/L, from about 50 mg/L to about 300 mg/L, from about 50 mg/L to about 250 mg/L, from about 50 mg/L to about 200 mg/L, from 50 mg/L to about 175 mg/L, from about 50 mg/L to about 150 mg/L, from about 50 mg/L to about 125 mg/L, or from about 50 mg/L to about 100 mg/L.

5.8.1 Further Methods of Engineering Antibodies

In another embodiment, an Fc hinge region of an antibody of the invention is mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, an antibody is modified to increase its biological half life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375. In another embodiment, one or more of the following mutations can be introduced: M252Y, S254T, T256E, as described in U.S. Pat. No. 7,083,784. Alternatively, to increase the biological half life, the antibody can be modified within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In other embodiments, an Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to reduce the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has reduced affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is reduced can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has reduced C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are modified to thereby reduce the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In yet another embodiment, an Fc region of an antibody of the invention is further modified to decrease the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta.

Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. In certain instances, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Thus, in another aspect of the invention, the structural features of anti-IFNAR1 antibodies, for example, but not limited to 3F11, 4G5, 11E2, and 9D4, are used to create structurally related anti-IFNAR1 antibodies that retain at least one functional property of antibodies of the invention, such as binding to IFNAR1. For example, one or more CDR regions of 3F11, 4G5, 11E2, or 9D4, or mutations thereof, can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-IFNAR1 antibodies of the invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the V_(H) and/or V_(L) sequences provided herein, or one or more CDR regions thereof. To create the engineered antibody, it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the V_(H) and/or V_(L) sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a “second generation” sequence(s) derived from the original sequence(s) and then the “second generation” sequence(s) is prepared and expressed as a protein.

5.9 Compositions

In another aspect, the present invention provides compositions containing one or a combination of monoclonal antibodies, or fusion proteins comprising an Fc region thereof, as described herein, formulated together with a carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, fusion proteins, immunoconjugates or bispecific molecules of the invention. In some embodiments, such compositions are physiologically tolerable and as such are suitable for administration to a subject (also referred to as a “pharmaceutical composition of the invention.” For example, pharmaceutical compositions of the invention may comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities.

In another embodiment, compositions of the invention may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

In another embodiment, compositions of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in contemplated compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

In another embodiment, compositions of the invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be suitable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization micro filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, compositions (e.g., liquid formulations) of the invention are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin. In one embodiment, endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. In another embodiment, endotoxin and pyrogen levels in the composition are less then about 10 EU/mg, or less then about 5 EU/mg, or less then about 1 EU/mg, or less then about 0.1 EU/mg, or less then about 0.01 EU/mg, or less then about 0.001 EU/mg.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, also from about 0.1 percent to about 70 percent, also from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

For administration of an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Dosage regimens for an anti-IFNAR1 antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

Alternatively, an antibody of fusion protein may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and usually until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A therapeutically effective dosage of an anti-IFNAR1 antibody of the invention results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. In the case of, for example, Systemic Lupus Erythematosus (SLE), a therapeutically effective dose may prevent further deterioration of physical symptoms associated with SLE, such as, for example, pain, fatigue or weakness. A therapeutically effective dose may also prevent or delays onset of SLE, such as may be desired when early or preliminary signs of the disease are present. Likewise it includes delaying chronic progression associated with SLE. Laboratory tests utilized in the diagnosis of SLE include chemistries, hematology, serology and radiology. Accordingly, any clinical or biochemical assay that monitors any of the foregoing may be used to determine whether a particular treatment is a therapeutically effective dose for treating SLE. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

A composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Selected routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in another embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

5.10 Diagnostic Uses

In other embodiments, antibodies of the present invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders.

In one embodiment, antibodies of the invention can be used to detect levels of IFNAR1, or levels of cells that express IFNAR1. This can be achieved, for example, by contacting a sample (such as an in vitro sample) and a control sample with the anti-IFNAR1 antibody under conditions that allow for the formation of a complex between the antibody and IFNAR1. Any complexes formed between the antibody and IFNAR1 are detected and compared in the sample and the control. For example, standard detection methods, well-known in the art, such as ELISA and flow cytometic assays, can be performed using the compositions of the invention.

Accordingly, in one aspect, the invention further provides methods for detecting the presence of IFNAR1 (e.g., human IFNAR1 antigen) in a sample, or measuring the amount of IFNAR1, comprising contacting the sample, and a control sample, with antibodies of the invention, or an antigen binding portion thereof, which specifically binds to IFNAR1, under conditions that allow for formation of a complex between the antibody or portion thereof and IFNAR1. The formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of IFNAR1 in the sample.

5.11 Therapeutic Applications

IFNAR1 is part of the cellular receptor for Type I interferons, and Type I interferons are known to be immunoregulatory cytokines that are involved in T cell differentiation, antibody production and activity and survival of memory T cells. Moreover, increased expression of Type I interferons has been described in numerous autoimmune diseases, in HIV infection, in transplant rejection and in graft versus host disease (GVHD). Accordingly, the anti-IFNAR1 antibodies of the invention or fragments thereof, which inhibit the functional activity of Type I interferons, can be used in a variety of clinical indications involving aberrant or undesired Type I interferon activity. The invention encompasses methods of preventing, treating, maintaining, ameliorating, or inhibiting a Type I interferon-mediated disease or disorder, wherein the methods comprise administering antibodies, or antigen-binding portions thereof, of the invention.

Specific examples of autoimmune conditions in which antibodies of the invention can be used include, but are not limited to, the following: systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's Disease, Ulcerative Colitis and Celiac's Disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis. Furthermore, the antibody compositions of the invention can be used for inhibiting or preventing transplant rejection or in the treatment of graft versus host disease (GVHD) or in the treatment of HIV infection/AIDS.

High levels of IFNα have been observed in the serum of patients with systemic lupus erythematosus (SLE) (see e.g., Kim et al. (1987) Clin. Exp. Immunol. 70:562-569). Moreover, administration of IFNα, for example in the treatment of cancer or viral infections, has been shown to induce SLE (Garcia-Porrua et al. (1998) Clin. Exp. Rheumatol. 16:107-108). Accordingly, in another embodiment, anti-IFNAR1 antibodies of the invention can be used in the treatment of SLE by administering the antibody to a subject in need of treatment.

Other methods of treating SLE are described in U.S. Patent Applications entitled “Methods of treating SLE” with the following serial numbers; 60/907,767, filed Apr. 16, 2007; 60/966,174, filed Nov. 5, 2007 and PCT application serial number PCT/US2007/02494, filed Dec. 9, 2007 each of which are incorporated by reference in their entireties.

IFNα also has been implicated in the pathology of Type I diabetes. For example, the presence of immunoreactive IFNα in pancreatic beta cells of Type I diabetes patients has been reported (Foulis et al. (1987) Lancet 2:1423-1427). Prolonged use of IFNα in anti-viral therapy also has been shown to induce Type I diabetes (Waguri et al. (1994) Diabetes Res. Clin. Pract. 23:33-36). Accordingly, in another embodiment, the anti-IFNAR1 antibodies or fragments thereof of the invention can be used in the treatment of Type I diabetes by administering the antibody to a subject in need of treatment. The antibody can be used alone or in combination with other anti-diabetic agents, such as insulin.

Antibodies to IFNAR1 have been shown to be effective in an animal model of inflammatory bowel disease (see U.S. Patent Application 60/465,155). Thus, the anti-IFNAR1 antibodies or fragments thereof of the invention can be used in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, by administering the antibody to a subject in need of treatment.

Treatment with IFNα has also been observed to induce autoimmune thyroiditis (Monzani et al. (2004) Clin. Exp. Med. 3:199-210; Prummel and Laurberg (2003) Thyroid 13:547-551). Accordingly, in another embodiment, anti-IFNAR1 antibodies of the invention can be used in the treatment of autoimmune thyroid disease, including autoimmune primary hypothyroidism, Graves Disease, Hashimoto's thyroiditis and destructive thyroiditis with hypothyroidism, by administering an antibody of the invention to a subject in need of treatment. Antibodies of the invention can be used alone or in combination with other agents or treatments, such as anti-thyroid drugs, radioactive iodine and subtotal thyroidectomy.

High levels of IFNα also have been observed in the circulation of patients with HIV infection and its presence is a predictive marker of AIDS progression (DeStefano et al. (1982) J. Infec. Disease 146:451; Vadhan-Raj et al. (1986) Cancer Res. 46:417). Thus, in another embodiment, anti-IFNAR1 antibodies of the invention may be used in the treatment of HIV infection or AIDS by administering the antibody of the invention to a subject in need of treatment. In another embodiment, antibodies of the invention can be used alone or in combination with other anti-HIV agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors.

Antibodies to IFNAR1 have been demonstrated to be effective in inhibiting allograft rejection and prolonging allograft survival (see e.g., Tovey et al. (1996) J. Leukoc. Biol. 59:512-517; Benizri et al. (1998) J. Interferon Cytokine Res. 18:273-284). Accordingly, the anti-IFNAR1 antibodies of the invention also can be used in transplant recipients to inhibit allograft rejection and/or prolong allograft survival. The invention provides a method of inhibiting transplant rejection by administering anti-IFNAR1 antibodies of the invention to a transplant recipient in need of treatment. Examples of tissue transplants that can be treated include, but are not limited to, liver, lung, kidney, heat, small bowel, and pancreatic islet cells, as well as the treatment of graft versus host disease (GVHD). Antibodies of the invention can be used alone or in combination with other agents for inhibiting transplant rejection, such as immunosuppressive agents (e.g., cyclosporine, azathioprine, methylprednisolone, prednisolone, prednisone, mycophenolate mofetil, sirilimus, rapamycin, tacrolimus), anti-infective agents (e.g., acyclovir, clotrimazole, ganciclovir, nystatin, trimethoprimsulfarnethoxazole), diuretics (e.g., bumetanide, furosemide, metolazone) and ulcer medications (e.g., cimetidine, farnotidine, lansoprazole, omeprazole, ranitidine, sucralfate).

In other specific embodiments, the invention provides methods of administering and using compositions and antibodies of the invention to treat and prevent a wide range of inflammatory conditions including both chronic and acute conditions, such as, but not limited to, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, herpes infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, celiac disease, congestive heart failure, restenosis, COPD adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fascitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, Retier's syndrome, and Hodgkins disease.

In another embodiment, methods of administration and compositions of antibodies of the invention may be useful in the prevention, treatment, amelioration of symptoms associated with the following conditions or disease states: Graves's disease, Hashimoto's thyroiditis, Crohn's disease, psoriasis, psoriatic arthritis, sympathetic opthalmitis, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome. Sjogren's syndrome, scleroderma, Addison's disease, polyendocrine deficiency syndrome, Guillan-Barre syndrome, immune thrombocytopenic purpura, pernicious anemia, myasthenia gravis, primary biliary cirrhosis, mixed connective tissue disease, vitiligo, autoimmune uveitis, autoimmune hemolytic anemia, autoimmune thrombopocytopenia, celiac disease, dermatitis herpetiformis, autoimmune hepatitis, pemphigus, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, autoimmune myocarditis, autoimmune vasculitis, alopecia areata, autoimmune artherosclerosis, Behcet's disease, autoimmune myelopathy, autoimmune hemophelia, autoimmune interstitial cystitis, autoimmune diabetes isipidus, autoimmune endometriosis, relapsing polychondritis, ankylosing spondylitis, autoimmune urticaria, dermatomyositis, Miller-Fisher syndrome, IgA nephropathy, goodpastures syndrome, and herpes gestationis.

In another embodiment, methods of administration and compositions of antibodies of the invention may be useful in the prevention, treatment, amelioration of symptoms associated with Sjogren's syndrome. Sjögren's syndrome is an autoimmune disorder in which immune cells attack and destroy the exocrine glands that produce tears and saliva. It is named after Swedish ophthalmologist Henrik Sjögren (1899-1986), who first described it. Sjögren's syndrome is also associated with rheumatic disorders such as rheumatoid arthritis, and it is rheumatoid factor positive in 90 percent of cases. The hallmark symptoms of the disorder are dry mouth and dry eyes. In addition, Sjögren's syndrome may cause skin, nose, and vaginal dryness, and may affect other organs of the body, including the kidneys, blood vessels, lungs, liver, pancreas, and brain. Nine out often Sjögren's patients are women and the average age of onset is late 40s, although Sjögren's occurs in all age groups in both women and men. It is estimated to strike as many as 4 million people in the United States alone making it the second most common autoimmune rheumatic disease.

Myositis is general condition characterized by inflammation of skeletal muscle or voluntary muscle. Muscle inflammation may be caused by an allergic reaction, exposure to a toxic substance or medicine, another disease such as cancer or rheumatoid conditions, or a virus or other infectious agent. The chronic inflammatory myopathies are idiopathic, meaning they have no known cause. They are understood to be autoimmune disorders, in which the body's white blood cells (that normally fight disease) attack blood vessels, normal muscle fibers, and connective tissue in organs, bones, and joints.

Polymyositis affects skeletal muscles (involved with making movement) on both sides of the body. It is rarely seen in persons under age 18; most cases are in patients between the ages of 31 and 60. In addition to symptoms listed above, progressive muscle weakness leads to difficulty swallowing, speaking, rising from a sitting position, climbing stairs, lifting objects, or reaching overhead. Patients with polymyositis may also experience arthritis, shortness of breath, and heart arrhythmias.

Dermatomyositis is characterized by a skin rash that precedes or accompanies progressive muscle weakness. The rash looks patchy, with bluish-purple or red discolorations, and characteristically develops on the eyelids and on muscles used to extend or straighten joints, including knuckles, elbows, heels, and toes. Red rashes may also occur on the face, neck, shoulders, upper chest, back, and other locations, and there may be swelling in the affected areas. The rash sometimes occurs without obvious muscle involvement. Adults with dermatomyositis may experience weight loss or a low-grade fever, have inflamed lungs, and be sensitive to light. Adult dermatomyositis, unlike polymyositis, may accompany tumors of the breast, lung, female genitalia, or bowel. Children and adults with dermatomyositis may develop calcium deposits, which appear as hard bumps under the skin or in the muscle (called calcinosis). Calcinosis most often occurs 1-3 years after disease onset but may occur many years later. These deposits are seen more often in childhood dermatomyositis than in dermatomyositis that begins in adults. Dermatomyositis may be associated with collagen-vascular or autoimmune diseases.

Inclusion body myositis (IBM) is characterized by progressive muscle weakness and wasting. IBM is similar to polymyositis but has its own distinctive features. The onset of muscle weakness is generally gradual (over months or years) and affects both proximal and distal muscles. Muscle weakness may affect only one side of the body. Small holes called vacuoles are seen in the cells of affected muscle fibers. Falling and tripping are usually the first noticeable symptoms of IBM. For some patients the disorder begins with weakness in the wrists and fingers that causes difficulty with pinching, buttoning, and gripping objects. There may be weakness of the wrist and finger muscles and atrophy (thinning or loss of muscle bulk) of the forearm muscles and quadricep muscles in the legs. Difficulty swallowing occurs in approximately half of IBM cases. Symptoms of the disease usually begin after the age of 50, although the disease can occur earlier. Unlike polymyositis and dermatomyositis, IBM occurs more frequently in men than in women.

Juvenile myositis has some similarities to adult dermatomyositis and polymyositis. It typically affects children ages 2 to 15 years, with symptoms that include proximal muscle weakness and inflammation, edema (an abnormal collection of fluids within body tissues that causes swelling), muscle pain, fatigue, skin rashes, abdominal pain, fever, and contractures (chronic shortening of muscles or tendons around joints, caused by inflammation in the muscle tendons, which prevents the joints from moving freely). Children with juvenile myositis may also have difficulty swallowing and breathing, and the heart may be affected. Approximately 20 to 30 percent of children with juvenile dermatomyositis develop calcinosis. Juvenile patients may not show higher than normal levels of the muscle enzyme creatine kinase in their blood but have higher than normal levels of other muscle enzymes.

Accordingly, in other embodiments, antibodies of the invention may be useful in the prevention, treatment, or amelioration of myositis, inflammatory myositis, idiopathic myositis, polymyositis, dermatomyositis, inclusion body myositis (IBM), juvenile myositis or symptoms associated with these conditions.

In another embodiment, antibodies of the invention may be useful in the prevention, treatment, or amelioration of symptoms associated with vasculitis.

Antibodies of the invention may be useful for the treatment of scleroderma. Methods of treating Scleroderma are described in a U.S. patent application entitled “Methods Of Treating Scleroderma” with an application serial number of 60/996,175, filed on Nov. 5, 2007 and PCT Application No. PCT/US2008/82481 are incorporated by reference in their entireties for all purposes.

In another embodiment, antibodies of the invention may be useful in the prevention, treatment, or amelioration of symptoms associated with sarcoidosis. Sarcoidosis (also called sarcoid or Besnier-Boeck disease) is an immune system disorder characterized by non-necrotizing granulomas (small inflammatory nodules). Virtually any organ can be affected; however, granulomas most often appear in the lungs or the lymph nodes. Symptoms can occasionally appear suddenly but usually appear gradually. When viewing X-rays of the lungs, sarcoidosis can have the appearance of tuberculosis or lymphoma.

Also within the scope of the invention are kits comprising the compositions (e.g., anti-IFNAR1 antibodies) of the invention and instructions for use. The kit can further contain a least one additional reagent, or one or more additional antibodies of the invention (e.g., an antibody having a complementary activity which binds to an epitope on the target antigen distinct from the first antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

5.12 Combinations

Compositions of the invention also can be administered in combination therapy, such as, combined with other agents. For example, the combination therapy can include an anti-IFNAR1 antibody of the present invention combined with at least one other immunosuppressent.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. The antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/ml and in some methods about 25-300 μg/ml.

When antibodies to IFNAR1 are administered together with another agent, the two can be administered in either order or simultaneously. For example, an anti-IFNAR1 antibody of the invention can be used in combination with one or more of the following agents: drugs containing mesalamine (including sulfasalazine and other agents containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide), non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g., predinisone, hydrocortisone), TNF-inhibitors (including adalimumab (HUMIRA®), etanercept (ENBREL®) and infliximab (REMICADE®)), immunosuppressants (such as 6-mercaptopurine, azathioprine and cyclosporine A), and antibiotics anti-IFNα antibody, anti-IFNγ receptor antibody, and soluble IFNγ receptor. Furthermore, an anti-IFNAR1 antibody of invention can be used in combination with a Flt3 ligand antagonist (see e.g., U.S. Application No. 2002/0160974).

In other embodiments, the compositions of the invention may also include agents useful in the treatment of SLE. Such agents include analgesics, corticosteroids (e.g., predinisone, hydrocortisone), immunosuppressants (such as cyclophosphamide, azathioprine, and methotrexate), antimalarials (such as hydroxychloroquine) and biologic drugs that inhibit the production of dsDNA antibodies (e.g., LJP 394).

5.13 Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to embodiments of the invention described herein.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

In addition, the following United States provisional patent applications: 61/006,962 filed Feb. 8, 2008, 61/034,618 filed Mar. 7, 2007, and 61/049,970 filed May 2, 2008 are hereby incorporated by reference herein in its entirety for all purposes.

5.14 Specific Embodiments

-   1. A modified IgG class monoclonal antibody specific for IFNAR1,     wherein said antibody comprises in the Fc region at least one amino     acid substitution selected from the group consisting of L234F,     L235E, and P331S, as numbered by the EU index as set forth in Kabat     and wherein said antibody exhibits reduced affinity for at least one     Fc ligand compared to an unmodified antibody. -   2. The antibody of embodiment 1, wherein, said antibody is an IgG1     or IgG4 subclass. -   3. The antibody of embodiment 2, wherein said antibody is an IgG1     class molecule. -   4. The antibody of embodiment 3, wherein said antibody comprises an     amino acid substitution of P331 S. -   5. The antibody of embodiment 3, wherein said antibody comprises the     amino acid substitutions: L234F and L235E. -   6. The antibody of embodiment 3, wherein said antibody comprises the     amino acid substitutions: L234F, L235E and P331S. -   7. The antibody of embodiment 3 wherein, said antibody is an IgG4     class molecule. -   8. The antibody of embodiment 7 wherein, said antibody comprises an     amino acid substitution of L235E of the Fc region. -   9. The antibody of embodiment 7, wherein, said antibody further     comprises in the Fc region amino acid substitution S228P. -   10. The antibody of any of embodiments 1-9 wherein, said antibody     comprises at least one complementarity determining region (CDR)     selected from Table 2. -   11. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region CDR1 comprising Seq ID         NO: 31;     -   b. a human heavy chain variable region CDR2 comprising Seq ID         NO: 32;     -   c. a human heavy chain variable region CDR3 comprising Seq ID         NO: 33;     -   d. a human light chain variable region CDR1 comprising Seq ID         NO: 34;     -   e. a human light chain variable region CDR2 comprising Seq ID         NO: 35; and     -   f. a human light chain variable region CDR3 comprising Seq ID         NO: 36. -   12. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region CDR1 comprising Seq ID         NO: 1;     -   b. a human heavy chain variable region CDR2 comprising Seq ID         NO: 2;     -   c. a human heavy chain variable region CDR3 comprising Seq ID         NO: 3;     -   d. a human light chain variable region CDR1 comprising Seq ID         NO: 4;     -   e. a human light chain variable region CDR2 comprising Seq ID         NO: 5; and     -   f. a human light chain variable region CDR3 comprising Seq ID         NO: 6. -   13. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region CDR1 comprising Seq ID         NO: 11;     -   b. a human heavy chain variable region CDR2 comprising Seq ID         NO: 12;     -   c. a human heavy chain variable region CDR3 comprising Seq ID         NO: 13;     -   d. a human light chain variable region CDR1 comprising Seq ID         NO: 14;     -   e. a human light chain variable region CDR2 comprising Seq ID         NO: 15; and     -   f. a human light chain variable region CDR3 comprising Seq ID         NO: 16. -   14. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region CDR1 comprising Seq ID         NO: 21;     -   b. a human heavy chain variable region CDR2 comprising Seq ID         NO: 22;     -   c. a human heavy chain variable region CDR3 comprising Seq ID         NO: 23;     -   d. a human light chain variable region CDR1 comprising Seq ID         NO: 24;     -   e. a human light chain variable region CDR2 comprising Seq ID         NO: 25; and     -   f. a human light chain variable region CDR3 comprising Seq ID         NO: 26. -   15. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region comprising the amino acid         sequence of Seq ID No: 38; and     -   b. a human light chain variable region comprising the amino acid         sequence of Seq ID No: 40. -   16. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region comprising the amino acid         sequence of Seq ID No: 8; and     -   b. a human light chain variable region comprising the amino acid         sequence of Seq ID No: 10. -   17. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region comprising the amino acid         sequence of Seq ID No: 18; and     -   b. a human light chain variable region comprising the amino acid         sequence of Seq ID No: 20. -   18. The antibody of any of embodiments 1-10, wherein, said antibody     comprises:     -   a. a human heavy chain variable region comprising the amino acid         sequence of Seq ID No: 28; and     -   b. a human light chain variable region comprising the amino acid         sequence of Seq ID No: 30. -   19. The antibody of any of embodiments 1-18, wherein, said antibody     comprises the light chain constant region sequence of Seq ID No: 41. -   20. The antibody of any of embodiments 1-18, wherein, said antibody     comprises the heavy chain constant region of Seq ID No: 42. -   21. The antibody of any of embodiments 1-18, wherein, said antibody     comprises the light chain constant region having the amino acid     sequence of SEQ ID No:41 and the heavy chain constant region having     the amino acid sequence of Seq ID No: 42. -   22. The antibody of any of embodiments 19-21, wherein, said antibody     comprises a heavy chain amino acid sequence comprising allelic     variation, wherein said allelic variation is at least one or more     positions selected from the group consisting of 214, 221, 356 and     358 as defined by the EU index numbering system. -   23. The antibody of any of the preceding embodiments wherein, said     antibody is selected from the group consisting of: human antibody,     humanized antibody, chimeric antibody, intrabody, and a synthetic     antibody. -   24. An isolated nucleic acid comprising a polynucleotide sequence     encoding the antibody of any of the preceding embodiments. -   25. The nucleic acid of embodiment 24 wherein, said nucleic acid is     a replicable vector. -   26. The nucleic acid of embodiment 25 wherein, said polynucleotide     sequence is operably linked to a promoter. -   27. A host cell comprising or transformed with the vector of     embodiment 25 or 26. -   28. A transgenic mouse comprising human immunoglobulin heavy and     light chain transgenes, wherein the mouse expresses the antibody of     any of embodiments 1-23. -   29. A hybridoma prepared from the mouse of embodiment 28 wherein the     hybridoma produces said antibody. -   30. A pharmaceutical composition comprising the antibody of any of     the embodiments 1-23, and a pharmaceutically acceptable excipient. -   31. A method of treating a condition or a disease associated with an     immune disorder, comprising administering to a subject in need     thereof an effective amount of the composition of embodiment 30. -   32. The method of embodiment 31 wherein said disease is a type I     interferon mediated disease. -   33. The method of embodiment 32 wherein said type I interferon is     interferon alpha. -   34. The method of embodiment 33 wherein said type I interferon     mediated disease is associated with the type I interferon receptor. -   35. The method of embodiment 31, wherein said disease or disorder is     HIV infection of AIDS. -   36. The method of embodiment 31, wherein said disease or disorder is     systemic lupus erythematosus. -   37. The method of embodiment 31, wherein said disease or disorder is     Sjogren's syndrome. -   38. The method of embodiment 31, wherein said disease or disorder is     myositis. -   39. The method of embodiment 31, wherein said disease or disorder is     inflammatory myositis. -   40. The method of embodiment 31, wherein said disease or disorder is     polymyositis. -   41. The method of embodiment 31, wherein said disease or disorder is     dermatomyositis. -   42. The method of embodiment 31, wherein said disease or disorder is     inclusion body myositis. -   43. The method of embodiment 31, wherein said disease or disorder is     juvenile myositis. -   44. The method of embodiment 31, wherein said disease or disorder is     idiopathic inflammatory myositis. -   45. The method of embodiment 31, wherein said disease or disorder is     vasculitis. -   46. The method of embodiment 31, wherein said disease or disorder is     sarcoidosis. -   47. The method of embodiment 31, wherein said disease or disorder is     selected from the group consisting of: inflammatory bowel disease,     multiple sclerosis, autoimmune thyroiditis, rheumatoid arthritis,     insulin dependent diabetes mellitus, glomerulonephritis, and graft     versus host disease. -   48. The method of embodiment 31, wherein said disease or disorder is     psoriasis or conditions resulting thereof. -   49. The method of embodiment 31, wherein said disease or disorder is     transplant rejection or graft versus host disease. -   50. The method of embodiment 31 wherein said disease or disorder is     selected from the group consisting of: Grave's disease, Hashimoto's     thyroiditis, Crohn's disease, psoriasis, psoriatic arthritis,     sympathetic opthalmitis, autoimmune oophoritis, autoimmune orchitis,     autoimmune lymphoproliferative syndrome, antiphospholipid syndrome.     Sjogren's syndrome, scleroderma, Addison's disease, polyendocrine     deficiency syndrome, Guillan-Barre syndrome, immune thrombocytopenic     purpura, pernicious anemia, myasthenia gravis, primary biliary     cirrhosis, mixed connective tissue disease, vitiligo, autoimmune     uveitis, autoimmune hemolytic anemia, autoimmune thrombopocytopenia,     celiac disease, dermatitis herpetiformis, autoimmune hepatitis,     pemphigus, pemphigus vulgaris, pemphigus foliaceus, bullous     pemphigoid, autoimmune myocarditis, autoimmune vasculitis, alopecia     areata, autoimmune artherosclerosis, Behcet's disease, autoimmune     myelopathy, autoimmune hemophelia, autoimmune interstitial cystitis,     autoimmune diabetes isipidus, autoimmune endometriosis, relapsing     polychondritis, ankylosing spondylitis, autoimmune urticaria,     dermatomyositis, Miller-Fisher syndrome, IgA nephropathy,     goodpastures syndrome, and herpes gestationis. -   51. The method of any of embodiments 31-50, further comprising     administering at least one agent selected from the group consisting     of: phototherapy, corticosteroids, prednisone, NSAIDS,     plasmapheresis, immunosuppressants, methotrexate, retinoic acid,     tioguanine, mycophenolate mofetil, fumaric esters, cyclophosphamide,     azathioprine, cyclosporine, and immunoglobulins. -   52. The method of any of embodiments 31-51 further comprising     administering at least one agent selected from the group consisting     of: alefacept (AMEVIVE™), etanercept (ENBREL®), adalimumab     (HUMIRA®), infliximab (REMICADE®), belimumab (LYMPHOSTATB™),     rituxumab (RITUXAN®), and efalizumab (RAPTIVA®). -   53. A crystal comprising a human TgG Fc region, wherein the human     IgG Fc region comprises at least one amino acid substitution     selected from the group consisting of L234F, L235E, and P331S, as     numbered by the EU index as set forth in Kabat and wherein said     fragment exhibits reduced affinity for at least one Fc ligand     compared to an unmodified Fc region. -   54. The crystal of Embodiment 53, wherein the human IgG Fc region     comprises the amino acid substitutions L234F, L235E and P331S. -   55. The crystal of Embodiment 53, which is diffraction quality. -   56. The crystal of Embodiment 53, which is a native crystal. -   57. The crystal of Embodiment 53, which is characterized by an     orthorhombic unit cell of a=50.18±0.2 Å, b=147.30±0.2 Å, and     c=75.47±0.2 Å. -   58. The crystal of Embodiment 53, which has a space group of C222₁. -   59. A modified monoclonal antibody, wherein said antibody comprises     in the Fc region the amino acid substitutions L234F, L235E, and     P331S, as numbered by the EU index as set forth in Kabat and wherein     said antibody exhibits reduced affinity for at least one Fc ligand     compared to an unmodified antibody. -   60. A fusion protein comprising a modified Fc region, wherein said     Fc region comprises the amino acid substitutions L234F, L235E, and     P331S, as numbered by the EU index as set forth in Kabat and wherein     said Fc region exhibits reduced affinity for at least one Fc ligand     compared to an Fc region. -   61. A method of making the antibody of any of embodiments 1-23 or     59. -   62. The antibody of any of embodiments 1-23 or 59, wherein said     antibody is an internalizing antibody. -   63. The fusion protein of embodiment 60, wherein said fusion protein     is an internalizing fusion protein. -   64. The fusion protein of embodiment 63, wherein said fusion protein     specifically binds IFNAR1. -   65. The antibody of any of embodiments 1-23, 59, or 62, wherein said     antibody exhibits reduced or ablated antibody dependent     cell-mediated cytotoxicity (ADCC) as compared to said unmodified     antibody. -   66. The antibody of any of embodiments 1-23, 59, or 62, wherein said     antibody exhibits reduced or ablated complement mediated     cytotoxicity (CDC) as compared to said unmodified antibody. -   67. The antibody of any of embodiments 1-23, 59, or 62, wherein said     antibody exhibits reduced or ablated ADCC and CDC as compared to     said unmodified antibody.

5.15 Sequences

Light Chain Constant Region (SEQ ID No:41)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Heavy Chain Constant Region (SEQ ID No:42)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

6. EXAMPLES

The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

6.1 Example 1: IHC Profile of Multiple Anti-IFNAR1 Antibodies

Purpose:

To evaluate the IHC profile of anti-IFNAR1 antibodies on a diverse set of tissues.

Methods:

Immunohistochemistry techniques to study antibody binding characteristics are readily known in the art and for example, could be performed by isolating the desired cells or tissues and preparing them for microscopy by standard fixation and mounting techniques.

Mouse Macrophages: A cell suspension was spun down to form a loose pellet. The pellet was frozen in OCT freezing medium to form a block. Slide sections were cut to 5 microns thickness, soaked in acetone for 10 minutes and allowed to dry with dessicant overnight. Prior to use, the slides were dipped into 10% neutral buffered formalin for 10 sec and washed 3× in buffer (1×TBS with 0.01% Tween20).

Human Monocytes: A cell suspension was smeared/spotted directly onto slides. The slides were allowed to dry overnight and then soaked in Acetone for 10 min and allowed to air dry. Prior to use, slides were dipped into 10% neutral buffered formalin for 10 sees and washed 3× in buffer (1×TBS with 0.01% Tween20).

Human Cerebrum and Cardiac Tissue: Tissue samples from donors were frozen in OCT freezing medium to form a block. Slide sections were cut to 5 micron thickness, soaked in acetone for 10 minutes and allowed to dry with dessicant overnight. Prior to use, the slides were dipped into 10% neutral buffered formalin for 10 sec and washed 3× in buffer (1×TBS with 0.01% Tween 20).

Antibody labeling: Antibodies were conjugated to biotin by the following protocol. Approximately 500 μg of antibody was mixed with a 20 fold excess of biotin and incubated for 2 hours in the dark at 4° C. After the 2 hour incubation, the antibody/biotin mix was applied to a pre-equilibrated PD10 column with IX PBS. Subsequently, the biotin conjugated antibodies were concentrated to a desired concentration using an YM-30 Centricon concentration tube.

Slide staining: After washing in buffer, slides were treated to quench endogenous peroxidases by treatment with a solution of Glucose Oxidase (1 U/ml, Sigma G0543), B-D(+) Glucose (10 mM, Sigma G5250), Sodium Azide (1 mM, Sigma, S8032) for 1 hour at room temperature. Slides were then rinsed in wash buffer (1×TBS with 0.01% Tween 20). Slides were placed in a protein block solution (lx PBS pH7.2, 0.5% casein(N-Z amine, Sigma C0626), 1% BSA (Sigma A7906), 1.5% Normal Goat serum (Jackson Labs #005-000-001) for 30 min at room temperature. Biotinylated antibody (see above) was applied to the slides by dilution into the protein block solution. Incubation of the slides with the biotinylated antibody was performed at room temperature for 2 hours. Slides were rinsed 3× in wash buffer (1×TBS, 0.01% Tween 20). Antibody detection was performed using a Vectastain Kit (Vector Laboratories). Slides were washed and counterstained with hematoxylin. Slides were dehydrated and mounted with coverslips prior to viewing.

Results:

Presented in FIG. 6A are the results of an IHC analysis of Human cerebrum tissue stained with various anti-IFNAR1 and control antibodies. The antibodies MDX-1333 (75 μg/ml) and 4G5 (50 μg/ml) exhibited strong staining of the cerebrum tissue as exemplified by the brown/dark staining seen throughout the samples. Antibody 9D4 (50 μg/ml) did not stain the human cerebrum tissue sample as well as MDX-1333 and 4G5 as demonstrated by the reduced brown/dark staining throughout the sample. An IgG1 isotype control was included to demonstrate that binding specificity of the individual antibodies.

Presented in FIG. 6B are the results of an IHC analysis of monocytes stained with various anti-IFNAR1 and control antibodies. The antibodies MDX-1333 (50 and 20 μg/ml), 4G5 (50 μg/ml) and 9D4 (50 and 20 μg/ml) all exhibited prominent staining on human monocytes as demonstrated by the brown/dark staining of the samples. The isotype control antibody R3-47 (50 μg/ml) did not exhibit prominent staining on human monocytes. In addition, MDX-1333(50 μg/ml) did not stain purified mouse macrophages.

Conclusions:

In IHC study the anti-IFNAR1 antibody 9D4 exhibited a lower level of staining as compared to other anti-IFNAR1 antibodies such as MDX-1333 and 4G5.

6.2 Example 2: Generation of Antibody 9D4 TM

The modified anti-IFNAR1 antibody designated “9D4-TM” was generated through the following procedure;

Human γ1 Fc was cloned and engineered from human PBLs by first isolating total RNA, transcribing cDNA, and PCR amplifying the constant regions with gene-specific primers containing restriction sites Apa I and EcoRI for cloning into the mammalian vector PEE6. The triple mutant (TM) includes three amino acid changes in human IgG to decrease ADCC effector function (L234F, L235E, and P331S). TM was engineered using human IgG1 (KOL) as a template, and utilizing site-directed mutagenesis (QuickChange XL, Stratagene) to encode three residue changes in the Fc. Sequence of the mutagenic primers used to encode the L234F/L235E/P331S changes were as follows:

(SEQ ID NO: 43) MD1056 = 5′ cgtgcccagcacctgaaTtcGAggggggaccgtcagtcttc 3′ L234F, L235E forward (SEQ ID NO: 44) MD1057 = 5′ gaagactgacggtccccccTCgaAttcaggtgctgggcacg 3′ L234F, L235E reverse (SEQ ID NO: 45) MD1058 = 5′ ccaacaaagccctcccagccTccatcgagaaaaccatctcc 3′ P331S forward (SEQ ID NO: 46) MD1059 = 5′ ggagatggttttctcgatggAggctgggagggctttgttgg 3′ P331S reverse

Clones encoding the 9D4-TM antibody were sequenced to confirm the triple mutations, and resolved on the ABI3100 genetic analyzer.

6.3 Example 3: Generation of Antibody 9D4 DM

The modified anti-IFNAR1 antibody designated “9D4-DM” was generated through the following procedure;

Human γ4 Fc was cloned and engineered from human PBLs by first isolating total RNA, transcribing cDNA, and PCR amplifying the constant regions with gene-specific primers containing restriction sites Apa I and EcoRI for cloning into the mammalian vector PEE6.

The double mutant (DM) consists of two mutations in human IgG4 Fc: S228P and L235E. Mutagenic primers to encode DM include:

(SEQ ID NO: 47) MD1060 = 5′ ggtcccccatgcccaCcatgcccagcacctg 3′ hinge S228P forward (SEQ ID NO: 48) MD1061 = 5′ caggtgctgggcatgGtgggcatgggggacc 3′ hinge S228P reverse (SEQ ID NO: 49) MD1062 = 5′ ccagcacctgagttcGAggggggaccatcagtc 3′ IgG4 L234F, L235E forward (SEQ ID NO: 50) MD1063 = 5′ gactgatggtccccccTCgaactcaggtgctgg 3′ IgG4 L234F, L235E reverse

Clones encoding the 9D4-DM antibody were sequenced to confirm the encoded changes, and resolved on the ABI3100 genetic analyzer.

6.4 Example 4 Anti-IFNAR1 Antibodies Inhibit IFN Mediated STAT Phosphorylation

Purpose:

To establish the ability of the anti-IFNAR1 antibody 9D4-TM to inhibit IFN mediated STAT phosphorylation in peripheral blood mononuclear cells.

Methods:

Peripheral blood mononuclear cells were purified from healthy human donors using LSM media (MP Biomedical, Solon Ohio). PBMCs were quantified and seeded at 10⁶ cell per condition per well. Antibodies were added at 10 μg/mL to appropriate well and incubated at 37° C., 5% CO₂ for 10 minutes. After pre-incubation with antibodies, recombinant human IFNα2a (PBL Biomedical, Piscataway N.J.) or human plasmacytoid dendritic cell-derived IFN (see below for generation of PDCs derived type-I IFN supernatants) was added to appropriate wells at 100 or 500 IU/mL for 20 minutes. Cells were spun at 1200 rpm for 5 minutes and washed with sterile 1×PBS. After one additional spin, PBS was removed and cells were lysed using mammalian protein extraction reagent (Pierce, Rockford Ill.) supplemented with 300 μL of 1× phosphatase inhibitor cocktails 1 and 2 (Sigma, St. Louis Mo.) and 1× protease inhibitor (Roche Biomedical, Nutley N.J.). Lysates were incubated for 10 minutes on an orbital shaker to ensure complete lysis, transferred to microfuge tubes and spun at 14000 rpm to remove cellular debris. NuPAGE sample buffer (Invitrogen, Carlsbad Calif.) and dTT (Sigma, St. Louis Mo.) were added to lysates for a final concentration of 1× and all samples were denatured in a heat block at 100° C. for approximately 10 minutes. 15 μL of each sample was added to NuPage 10% Bis-tris polyacrylamide gel (Invitrogen, Carlsbad Calif.) in NuPAGE MES SDS running buffer supplemented with 1× NuPAGE antioxidant buffer. Samples were run at 180V for 30 minutes for separation of protein bands. Proteins were then transferred to a nitrocellulose membrane and blots were blocked with 1×PBS (Gibco BRL, Carlsbad Calif.) containing 5% BSA (Sigma, St. Louis Mo.) overnight at 4° C. Blocking media was subsequently removed and 0.2 μg/mL anti-STAT1, anti-STAT1 pY701, or 1:1000 dilution of β-Actin antibodies (Cell Signaling Technology, Danvers Mass.) were added to appropriate blots and incubated overnight at 4° C. Blots were washed 3× in 1×TBS with 0.05% Tween20 (Sigma, St. Louis Mo.). 1:2500 diluted, HRP conjugated anti-rabbit secondary antibody was added to blots and incubated for 1 hr at room temperature. Blots were washed as described before and 3 mL of a 1:1 mixture of Pico Supersignal West reagent (Pierce, Rockford Ill.) was added to each blot for 1 minute. Blots were drained, excess reagent was removed and bands were visualized using a Kodak X-omat 1000A Processor.

Results:

Presented in FIG. 7. are the results of a STAT activation assay in which cells stimulated with Leukocyte IFN in the presence or absence of anti-IFNAR1 antibodies. In the absence of antibodies, leukocyte interferon stimulates the phosphorylation of STAT isoforms 1, 3 and 5. Incubation of cells with 9D4-TM antibody inhibits the phosphorylation mediated by treatment with leukocyte interferon. Cells treated with the isotype control antibody R3-47 do not exhibit inhibition of STAT phosphorylation in response to stimulation with leukocyte interferon.

Conclusions:

The results in FIG. 7 demonstrate that 9D4-TM is capable of inhibiting responses to IFNα such as the induction of STAT phosphorylation in peripheral blood mononuclear cells.

6.5 Example 5: Anti-IFNAR1 Antibodies Inhibit Type I IFN Signaling

Purpose:

Using purified Type I IFN from pDC cells, a reporter assay was used to test the ability of anti-IFNAR1 antibodies to block Type I IFN signaling.

Methods:

Plasmacytoid dendritic cells (PDCs) were isolated from whole blood of healthy donors using a lymphocyte separation media (MP Biomedical, Solon Ohio) followed by positive selection using CD304 (BDCA-4/Neuropilin-1) MicroBead Kit (Milteny Biotec, Auburn Calif.). Purified PDCs were then cultured at 1×10⁶ cells/mL in RPMI 1640 supplemented with 10% FBS (Gibco BRL) and 6 μg/mL CpGA (InVivogen, San Diego Calif.). Supernatants were harvested and clarified after 20 hours in culture and type-I IFN was quantified using a stably transfected HEK293-ISRE reporter cell line against a standard curve of human leukocyte IFN (PBL Biomedical, Piscataway N.J.).

pDCs from three healthy human donors were used to generate human pDC-derived type-I interferon supernatants, as described above. HEK293 (ATCC, Manassas Va.) cells were stably transfected with pHTS-ISRE reported plasmid (Biomyx Technology, San Diego Calif.) and were maintained in DMEM supplemented with 10% FBS, 1×NEAA, and 700 μg/mL G418 (Invitrogen, Carlsbad Calif.). Cells were seeded at a concentration of 80,000 cells per well in Optilix white/clear 96 well plates (VWR, West Chester Pa.). Appropriate concentrations of antibodies (611-0.00004 nM) were added to each well followed by addition of appropriate concentrations of human PDC-derived type-I interferon supernatants. Cells, IFN, and antibodies were incubated overnight at 37° C., 5% CO₂ and amplification of the luciferase protein was evaluated by lysing the cells with Cell Culture Lysis reagent and visualization using the Luciferase Assay System (Promega, Madison Wis.). Signal was measured in cps and IC50 values were generated.

Results:

Type I IFN supernatants were harvested from pDC cells derived from three individual donors. In a Luciferase reporter assay, incubation of anti-IFNAR1 antibodies inhibited the signaling ability of various concentrations of Type I IFN supernatant (FIG. 8).

Conclusions:

These results demonstrate that anti-IFNAR1 antibodies are capable of inhibiting Type I IFN mediated signaling as measured by reporter assay activity.

6.6 Example 6: Modified Anti-IFNAR1 Antibodies Exhibit Similar Binding Characteristics to the Parental Unmodified Antibody

Purpose:

To investigate the IFNAR1 binding characteristics of modified antibodies as compared to parental unmodified versions. Represented in FIG. 9 are the binding affinity curves for 9D4, 9D4DM, and 9D4TM. The binding constants (Kd) for the 9D4, 9D4DM, and 9D4TM anti-IFNAR1 antibodies were determined from the binding curves.

Methods:

200,000 HEK 293F cells were seeded in a round bottom, 96-well plate using 50 μL RPMI 1640 media supplemented with 10% FBS. Europium-labeled 9D4-TM was prepared under contract by PerkinElmer Life and Analytical Sciences. To measure non-specific Europium signal, 25 μL of 100-fold excess unlabeled, serially diluted anti-IFNAR1 antibodies were added to appropriate wells of the 96 well for 5-10 minutes prior to the addition of labeled 9D4-TM. 25 μL of europium conjugated, serially diluted antibody was then added to appropriate wells and cells and antibodies were agitated gently at room temperature for 1-2 hours. After binding incubation, 150 μL of cell media was added to all wells and plates were spun at 1200 rpm for 5 minutes at room temperature. Plates were quickly decanted and 250 μL cell media was added to all wells. Spins and washes were repeated for a total of 3 washes. Cells were then resuspended in 100 μL cell media. 50 μL of resuspended cells were transferred to 200 μL of DELPHIA enhancement solution (PerkinElmer) in a DELPHIA yellow microtiter plate and Europium emission was measured on a Victor2 Multilabel reader (PerkinElmer). Signal was measured in cps and K_(d) values and B_(max) values were generated using GraphPad Prism 4 analysis software.

Results:

The data represented in FIG. 9 demonstrates that the modified antibodies 9D4-TM and 9D4-DM exhibit similar binding affinities for IFNAR1 (9D4=0.06+/−0.02 nM, 9D4-DM=0.06+/−0.02 nM, 9D4-TM=0.03+/−0.01 nM) to the parental unmodified antibody.

Conclusions:

The data presented in this example demonstrates that the modified antibodies share similar IFNAR1 binding characteristics with the parental unmodified antibodies.

6.7 Example 7: Equilibrium Binding Assay Data for 9D4-TM Vs. sIFNαRI

Purpose:

To determine equilibrium binding data for 9D4-TM using soluble IFNAR1 (srIFNAR1)

Methods:

srIFNAR1 ligand was coated onto UltraLink® Biosupport beads (PIERCE, Rockford, Ill.) at concentrations of 5 μg/mL and 50 μg/mL in coating buffer (50 mM sodium carbonate buffer, pH9) for a period of 1-2 days at 4° C. Coated beads were then separated (gentle pulse spin) from unreacted ligand solution, and gently rocked in block buffer (1 mL 1M Tris, pH8, containing BSA at 10 mg/mL) for about 15 min at room temperature (RT). After this, the bead slurry was again spun to remove the blocking solution, and then the block step was repeated for about 2 hrs at RT using a fresh aliquot of block buffer. Following the blocking step, the coated beads were stored at 4° C. until used. Prior to use, the srIFNAR1-coated beads were transferred to a bead vial, resuspended in 27 mLs of instrument run buffer (PBS, pH7.4-0.02% NaN3), then attached to the KinExA 3000 instrument.

All equilibrium binding constants (K_(D)) were obtained from measurements made on a KinExA 3000 instrument (Sapidyne Instruments, Boise, Id.). Briefly, 9D4-TM IgG was prepared at 1 pM, 10 pM and 50 pM and dispensed into three series of tubes. This range of IgG concentrations was designed to permit measurements to be made at under both receptor- and K_(D)-controlled conditions. Two-fold serial dilutions of srIFNAR1 ligand were then titrated across these IgG solutions, at concentrations ranging from 19.5 fM-1 nM. Based on the vendor-supplied, theory curve simulations available through the software (Sapidyne Instruments, Boise, Id.), these equilibration mixtures were incubated anywhere from 2-6 days at RT. At the end of this time, signal-testing experiments were conducted to determine the appropriate run conditions. Detection of free antibody was made possible using a species-specific, Cy5-labeled secondary antibody reagent (Cy5 AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, Part #109-176-097, Jackson ImmunoResearch Laboratories), employed at 0.1, 1.0 or 2.0 μg/mL of PBS, pH7.4-0.02% NaN3 containing BSA at 1 mg/mL. Data obtained from the experiments were then simultaneously fitted using the software provided n-Curve analysis feature to obtain the reported binding constant (K_(D)).

Results:

Depicted in FIG. 10A are the binding curves for three concentrations of 9D4-TM (1 pM, 10 pM, and 50 pM) with sIFNαRI. Data obtained from at least three independent experiments were fitted to a software derived binding curve to establish a relative K_(D) for 9D4-TM. The K_(D) of 9D4-TM in this binding assay was determined to be 1.1 pM with a 95% confidence interval of 0.603 pM-1.8 pM. The percentage error of the K_(D) determination of 1.1 pM was 1.96%. The Kon and Koff for 9D4-TM was also determined to be 7×10⁶+/−1.3×10⁶ S⁻¹ and 7.7×10⁻⁶+/−1.57×10⁻⁶ l/Ms respectively (data not shown).

Conclusions:

The modified anti-IFNAR1 antibody 9D4=TM exhibits a very low K_(D) of approximately 1.1 pM, for sIFNAR1 as determined by the KinExa assay.

6.8 Example 8: Determination of Binding Affinity of 9D4-TM on Human PBMCs

Purpose:

To determine the binding affinity on human PBMC's

Methods:

Peripheral blood mononuclear cells were purified from healthy human donors using LSM media (MP Biomedical, Solon Ohio). Cells were counted and 200,000 cells were seeded in a round bottom, 96-well plate using 50 μL RPMI 1640 media supplemented with 10% FBS. Europium-labeled 9D4-TM was prepared under contract by PerkinElmer Life and Analytical Sciences. To measure non-specific europium signal, 25 μL of 100-fold excess unlabeled, serially diluted 9D4-TM was added to appropriate wells of the 96 well for 5-10 minutes prior to the addition of labeled 9D4-TM. 25 μL of europium conjugated, serially diluted 9D4-TM was then added to appropriate wells and cells and antibodies were agitated gently at room temperature for 1-2 hours. After binding incubation, 150 μL of cell media was added to all wells and plates were spun at 1200 rpm for 5 minutes at room temperature. Plates were quickly decanted and 250 μL cell media was added to all wells. Spins and washes were repeated for a total of 3 washes. Cells were then resuspended in 100 μL cell media. 50 μL of resuspended cells were transferred to 200 μL of DELPHIA enhancement solution (PerkinElmer) in a DELPHIA yellow microtiter plate and Europium emission was measured on a Victor2 Multilabel reader (PerkinElmer). Signal was measured in cps and Kd values and B max values were generated using GraphPad Prism 4 analysis software.

Results:

Using the affinity measurements documented in FIG. 10B. it was determined that the Kd for 9D4-TM binding to human PBMCs was 0.29 nM+/−0.11 nM with the number of binding sites determined to be 1448+/−447. Using a similar approach, the affinity binding constant for cynomologus monkey IFNAR was determined to be 0.65+/−0.42 nM with the number of binding sites determined to be 648+/−204 (data not shown).

Conclusions:

The results presented in FIG. 10B demonstrate that 9D4-TM binds specifically and with high affinity to human PBMCs.

6.9 Example 9: The Modified Anti-IFNAR1 Antibodies Exhibit Similar Potency with the Parental Unmodified Antibody

Purpose:

To demonstrate that modified anti-IFNAR2 antibodies (i.e. anti-IFNAR1 antibodies with reduced Fc ligand affinity) exhibit similar potency with the parental unmodified antibodies.

Methods:

The Luciferase Reporter assay system used in this example has been previously described above (See Example 3). Antibodies to IFNAR1 used in this example include 9D4, 9D4-DM, 9D4-TM, MDX-1333. Included is a control antibody R3-47.

Results:

Using the Luciferase reporter system, 1C50 values were generated for the various anti-IFNAR1 antibodies described above (See FIG. 11A). The anti-IFNAR1 antibody 9D4 (0.01 nM) and the modified antibodies, such as 9D4-DM (0.01 nM) and 9D4-TM (0.02 nM) each elicit a similar IC50 value in the reporter assay demonstrating that they exhibit a similar potency. Another anti-IFNAR1 antibody, MDX1333 (0.04 nM) also exhibits a similar potency to the unmodified 9D4 antibody. The isotype control does not inhibit Type I IFN mediated signaling in this Luciferase reporter assay.

Conclusions:

Modified anti-IFNAR1 antibodies share similar potencies to the unmodified versions as demonstrated by IC50 values generated in a Luciferase Reporter assay system designed to quantify IFN signaling events.

6.10 Example 10: 9D4-TM Inhibits the Activity of Multiple Type I Interferon Alpha Isoforms

Purpose:

To demonstrate that 9D4-TM inhibits signaling attributed to specific and multiple interferon alpha isoforms.

Methods:

The Luciferase Reporter assay system used in this example has been previously described above (See Example 5). Results: The IC50 values for the 9D4-TM mediated inhibition of Type I interferon activity are presented in Table 4.

TABLE 4 IC50 values for 9D4-TM mediated inhibition of Type I interferon activity Type I Interferon 9D4-TM IC50 (nM) IFN-α2b 0.07 +/− 0.01 IFN-α2a 0.3 +/− 0.2 IFN-α6 0.04 +/− 0.01 IFN-α16 0.02 +/− 0.03 IFN-α8 0.03 +/− 0.04 IFN-α10 0.01 +/− 0.01 Leukocyte Interferon 0.01 +/− 0.01 IFN-α17 0.04 +/− 0.03 IFN-α14 0.02 +/− 0.01 IFN-α1 0.004 +/− 0.01  IFN-α21  0.01 +/− 0.002 IFN-α7 0.04 +/− 0.01 IFN-α4b 0.02 +/− 0.01 IFN-β1 6.8 +/− 9.4 IFN-ω 0.1 +/− 0 

As shown, 9D4-TM exhibits IC50 values in the sub-nanomolar range for multiple interferon alpha isoforms, leukocyte interferon, and interferon omega.

Conclusions:

The modified anti-IFNAR1 antibody 9D4-TM demonstrates the ability to inhibit the signaling attributed to multiple specific interferon alpha subtypes as well as leukocyte interferon alpha in a reporter assay

6.11 Example 11: Isoelectric Point Determination of 9D4, 9D4DM and 9D4TM

Purpose:

To evaluate the biophysical characteristics of the parental unmodified antibody 9D4 in comparison to the modified antibodies 9D4-DM and 9D4-TM.

Methods:

Native Isoelectric Focusing Polyacrylamide Gel Electrophoresis (IEF-PAGE) analysis was performed as follows: Pre-cast ampholine gels (Amersham Biosciences, pI range 3.5-9.5) were loaded with 8 μg of protein. Protein samples were dialyzed in 10 mM Histidine pH-6 before loading on the gel. Broad range pI marker standards (Amersham, pI range 3-10, 8 μL) were used to determine relative pI for the Mabs. Electrophoresis was performed at 1500 V, 50 mA for 105 minutes. The gel was fixed for 45 minutes using a Sigma fixing solution (5×) diluted with purified water to 1×. Staining was performed overnight at room temperature using Simply Blue stain (Invitrogen). Destaining was carried out with a solution that consisted of 25% ethanol, 8% acetic acid and 67% purified water. Isoelectric points were determined using a Bio-Rad GS-800 Densitometer with Quantity One Imaging Software.

Results:

Depicted in FIG. 12A is the isoelectric point (pI) determination for antibodies 9D4WT, 9D4DM, and 9D4TM. Samples of the antibodies were run according to the methods above and exhibited the following characteristics. The 9D4 WT antibody exhibited prominent protein bands corresponding to 8.2, 8.35 and 8.51. The 9D4 DM antibody exhibited a single prominent protein band corresponding to 7.13. The 9D4 TM antibody exhibited prominent protein bands corresponding to 8.09 and 8.18.

Conclusions:

As presented in this Example, the modified antibodies 9D4-DM and 9D4-TM exhibit very similar biophysical characteristics (pI) to the parental unmodified antibody 9D4.

6.12 Example 12: Thermostability of 9D4, 9D4-DM and 9D4-TM

Purpose:

To evaluate the biophysical characteristics of the parental unmodified antibody 9D4 in comparison to the modified antibodies 9D4-DM and 9D4-TM.

Methods:

Differential Scanning Calorimetry was performed as follows: thermal melting temperatures (T_(m)) were measured with a VP-DSC (MicroCal, LLC) using a scan rate of 1.0° C./min and a temperature range of 20-110° C. A filter period of 8 seconds was used along with a 15 minute pre-scan. Samples were prepared by dialysis into 10 mM Histidine-HCl, pH 6 using Pierce dialysis cassettes (3.5 kD). Mab concentrations were 0.14 mg/ml, 0.79 mg/ml, and 0.64 mg/ml as determined by A₂₈₀. Melting temperatures were determined following manufacturer procedures using Origin software supplied with the system. Briefly, multiple baselines were run with buffer in both the sample and reference cell to establish thermal equilibrium. After the baseline was subtracted from the sample thermogram, the data were concentration normalized.

Results:

The antibodies 9D4, 9D4-DM, 9D4-TM were subjected to differential scanning calorimetry as detailed above with the results presented in FIG. 12B. Each of the antibodies studied exhibited similar melting temperatures in the assay. Specifically, the antibodies exhibited the following melting temperatures; 9D4 WT=70.41° C., 9D4-DM=70.41° C., and 9D4-TM=70.88° C.

Conclusions:

As presented in this Example, the modified antibodies 9D4-DM and 9D4-TM exhibit very similar biophysical characteristics (T_(m)) to the parental unmodified antibody 9D4.

6.13 Example 13: Surrogate Anti-IFNAR Antibodies Protect Mice from IFNα Induced Proteinuria

Purpose:

To demonstrate that anti-IFNAR antibodies protect mice from induced proteinuria in a model of SLE.

Methods:

Female NZB/W F1 mice were purchased from Jackson Labs and housed in pathogen-free barrier facility. The recombinant adenovirus vector containing the mouse IFNα subtype 5 cDNA under the control of the CMV promoter/enhancer (Adv-mIFNα5) was used to induce early lupus in these mice. Mice (8 mice/group) were treated at 8-11 wk of age with a single i.v. injection of 0.3×10¹⁰ Adv-mIFNα5 viral particles (vp). Controls received the same amount of control Adv particles. In some experiments, mice were injected with gradual doses of Adv-mIFNα5 ranging from 0.01×10¹⁰ to 1.0×10¹⁰ vp/mouse. To test the efficacy of anti-IFNAR1, mice were treated with successive 5 daily i.p. dosing of antibody at 10 mg/kg starting at the time of Adv delivery. For proteinuria, urine was tested using a dipstick (Chemstrip 2 GP; Roche Diagnostics). Proteinuria scored as 1 for levels of 30 mg/dl, 2 for 100 mg/dl, and 3 for levels ≥500 mg/dl. Mice were considered to have proteinuria if two consecutive urine samples scored 2 or higher.

Results:

The results of the adenovirus infected mice treated with anti-IFNAR1 antibodies are presented in FIG. 13. Mice infected with Adv-mIFNα5 exhibit proteinuria with an onset of about 3 weeks. Infected mice treated with control mouse IgG antibody are not protected from the onset of proteinuria over the course of the experiment as demonstrated by an onset of proteinuria of about 4 weeks. Mice treated with anti-IFNAR antibodies do not show evidence of proteinuria throughout the 8 week time course. Mice treated with an adenovirus control show no proteinuria over the experimental time course.

Conclusions:

Taken together, the data in this example demonstrates that the presence of anti-IFNAR antibodies is protective against adv-IFN induced proteinuria in an in vivo mouse model.

6.14 Example 14: Anti-IFNAR Antibodies Block Type I IFN Induced Gene Regulation

Purpose:

To demonstrate that anti-IFNAR1 antibodies inhibit or reduce Type I interferon gene regulation in a mouse model of SLE.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. RNA was prepared from tissues using RLT lysis buffer (Qiagen). For solid tissues (kidney, spleen, skin), no more than 50 mg of tissue was used for RNA processing each time. Samples were placed in lysis buffer and lysing matrix A (Qbiogene), and processed for 30 sec at 4.5 m/s using Fastprep24 homogenizer instrument (Thermo Electron Corporation, Waltham, Mass.). For PBMC, whole blood samples were centrifuged and the pellet was lysed in RLT buffer. Upon lysis, samples were snap frozen at −80° C. until further processed. To isolate RNA, thawed tissue lysates were first processed using Qiashredder spin columns, then equal volumes of 70% ethanol were added to the tissue lysates and RNA was purified using Rneasy mini spin column kits according to the manufacturer's instruction.

cDNA was generated from 3 μg of RNA using SuperScript III reverse transcriptase and oligo d(T) as described in the manufacturer's protocol (Invitrogen, Corp. Carlsbad, Calif.). Samples of cDNA were diluted in nuclease-free water and stored at −80° C.

Expression levels of selected genes were measured by real-time PCR TaqMan® analysis using the ABI 7900HT Fast Real-time PCR system (Applied Biosystems, Foster City, Calif.). Housekeeping gene β-actin was used for endogenous control. Reaction mixtures had a final volume of 20 μl consisting of 1 μl of cDNA, 2 μl of 20× primers and probes (TaqMan® Gene Expression Assays, Applied Biosystems) and 18 μl of diluted TaqMan® Fast Universal PCR Master Mix. Amplification conditions were: 20 seconds at 95° C., 50 cycles of 1 second at 95° C. and 20 seconds at 60° C. CT values range from 0 to 50, with the latter number assumed to represent no product formation. Quantification of gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to the housekeeping gene.

Results:

Type I interferon ectopically expressed in mice (See example 13) leads to induction of a number of genes. Presented in FIG. 14 are the fold changes of six Type I interferon responsive genes in the different populations of mice used in this experiment. Specifically, genes IFIT1, IFI44, IFI202b, CXCL9, CXCL10, and CXCL11 are all induced in the mice ectopically expressing IFNα and treated with nonspecific Mouse IgG. Mice ectopically expressing IFNα and treated with anti-IFNAR antibodies do not show any induction of the six Type I interferon responsive genes. As a control to demonstrate specificity of the adenovirally encoded IFNα, mice treated with PBS, or control adenovirus do not show any induction of these 6 genes. These results demonstrate that the administration of anti-IFNAR antibodies can block the gene induction response to IFN alpha in an in vivo mouse model.

Conclusions:

Anti-IFNAR antibodies can block the regulation of Type I responsive genes in mouse model of SLE.

6.15 Example 15: Anti-IFNAR Antibodies Block the Production of Anti-dsDNA and Anti-SSA/Ro (Anti-Nuclear Antigen) Antibodies Induced by Type I Interferon

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to block the production of anti-nuclear antibodies, such as anti-dsDNA and anti-SSa/Ro induced by Type I interferon in a mouse model of SLE.

Methods:

Mice were prepared and treated as described in Example 13. Serum anti-dsDNA autoantibody levels were assessed by ELISA. Briefly, ELISA plates pretreated with poly (L-lysine) (100 μg/ml) were coated with calf thymus activated DNA (5 μg/ml in carbonate-bicarbonate buffer) (SIGMA). After overnight incubation at 4° C., plates were blocked with PBS/10% FCS. Sera (1/200 dilution) were incubated for 30 minutes at room temperature. Bound IgG was detected with peroxidase-conjugated goat anti-mouse IgG (1/4000) (KPL) added to the plates for 30 min. Binding was measured by adding TMB substrate (KPL) and stop solution (KPL), and the OD was read at 450 nm. A mouse anti-ds DNA IgG standard in serum was run in serial dilution (from 625 ng/ml) (Alpha Diagnostic) on each plate to allow standardization. Serum anti-SSA/Ro autoantibody levels were measured by ELISA (Alpha Diagnostic) following the manufacturer's instructions.

Results:

Type I interferon ectopically expressed in mice (See Example 13) leads to accumulation of anti-dsDNA and anti-SSA/Ro antibodies. Presented in FIG. 15 are the relative quantities of anti-dsDNA (A) and anti-SSA/Ro (B) antibodies in the different populations of mice (control adenovirus, Adv-IFNα+PBS, Adv-IFNα+MuIgG, and Adv-IFNα+Anti-IFNAR) as measured by ELISA. Control adenovirus infected mice show little accumulation of anti-dsDNA or anti-SSA/Ro antibodies in this experiment. Mice infected with adenovirus encoding IFNα and treated with PBS accumulate anti-dsDNA and anti-SSA/Ro antibodies. Adv-IFNα infected mice treated with anti-IFNAR antibodies acquire less anti-dsDNA and anti-SSA/Ro antibodies than Adv-IFNα infected mice treated with non-specific IgG. These results demonstrate that treatment with anti-IFNAR antibodies inhibits the accumulation of anti-dsDNA and anti-SSA/Ro antibodies in response to ectopically expressed Type I IFN.

6.16 Example 16: Anti-IFNAR Antibodies Block the Production of IP-10 and IL-18 Induced by Type I Interferon

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to block the accumulation of IFNα induced cytokines in a mouse model of SLE.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. Serum levels of cytokines were measured by ELISA (R&D systems) following the manufacturer's instructions.

Results:

Type I interferon ectopically expressed in mice (See Example 13) leads to accumulation of IP-10 and IL-18 cytokines. Presented in FIG. 16 are the relative quantities of IP-10 (A) and IL-18 (B) in the different populations of mice (PBS, control adenovirus, Adv-IFNα+MuIgG, and Adv-IFNα+Anti-IFNAR) as measured by ELISA. Type I interferon ectopically expressed in mice (See Example 12) leads to accumulation of the cytokines, IP-10 and IL-18. Control adenovirus infected mice show little accumulation of IP-10 (A) or IL-18 (B) cytokines in this experiment. Adv-IFNα infected mice treated with anti-IFNAR antibodies accumulate less IP-10 and IL-18 cytokines than Adv-IFNα infected mice treated with non-specific IgG. These results demonstrate that treatment with anti-IFNAR antibodies inhibits the accumulation of the cytokines IP-10 and IL-18 in response to ectopically expressed Type I IFN.

Conclusions:

Anti-IFNAR antibodies are able to block the accumulation of IFNα induced cytokines in a mouse model of SLE.

6.17 Example 17: Anti-IFNAR Antibodies Block the Production of ANA (Anti-Nuclear Antibodies) Induced by Type I Interferon

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to block the accumulation of IFNα induced anti-nuclear antibodies in a mouse model of SLE.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. Antinuclear antibody (ANA) levels were measured by ANA test kit (Antibodies Incorporated) with Hep-2 stabilized substrate and mitotic figures following the manufacturer's instruction. Serum was serially diluted and incubated with the Hep-2 cells on slides and the bound antinuclear antibody was detected by Hi-FITC labeled goat anti-mouse IgG (H+L) (Antibodies Incorporated). The titer of ANA is defined as the serum dilution factor where the ANA is no longer detectable.

Results:

Type I interferon ectopically expressed in mice (See Example 13) leads to accumulation of anti-ANA antibodies. Presented in FIG. 17 is the serum titer of anti-ANA antibodies in the different populations of mice (no virus, control adenovirus, Adv-IFNα+PBS, Adv-IFNα+MuIgG, and Adv-IFNα+Anti-IFNAR) as measured by serial dilution staining on HEP2 cells. Control adenovirus infected mice show little accumulation of anti-ANA antibodies in this experiment. Mice infected with adenovirus encoding IFNα and treated with PBS accumulate anti-ANA antibodies. Adv-IFNα infected mice treated with anti-IFNAR antibodies acquire less anti-ANA antibodies than Adv-IFNα infected mice treated with non-specific IgG. These results demonstrate that treatment with anti-IFNAR antibodies inhibits the accumulation of anti-ANA antibodies in response to ectopically expressed Type I IFN.

Conclusions:

Anti-IFNAR antibodies are able to block the accumulation of anti-nuclear antibodies induced by IFNα in a mouse model of SLE.

6.18 Example 18: Antibody Inhibition of SLE Plasma Mediated Dendritic Cell Development

Purpose:

SLE plasma induces dendritic cell development from normal human monocytes. In this example, the purified monoclonal antibody 9D4-TM was tested for the inhibition of dendritic cell development, as assessed by the ability of the antibodies to inhibit the induction of the cell surface markers CD38, and CD123 by SLE plasma.

Methods:

The methods have been previously described in US Patent Application No. 20006/0029601 and is hereby incorporated by reference in its entirety. Essentially, the experiments were conducted as follows: A 25 ml buffy coat was diluted four-fold with phosphate buffered saline (PBS). The sample was separated into 4×50 ml conical tubes, and 15 ml of lymphocyte separation medium (ICN Biomedicals) was layered underneath. After a 30 minute spin at 500 g, the buffy layer containing the peripheral blood mononuclear cells (PBMCs) was removed and washed with PBS. Cells were resuspended in culture media containing 1% heat inactivated human serum at 4×10⁶ cells/ml. Monocytes were isolated by incubating PBMCs (2.0×10⁷ cells/5 ml/25 cm² flask) for 1.5 hours at 37° C. in culture media and then washing away non-adherent cells twice. For induction of monocyte maturation, the cells were incubated with medium containing 25% human plasma from healthy volunteers or from patients with SLE. Antibody blocking studies were conducted by adding 30 μg/ml of anti-IFNAR1 antibody or isotype control, IgG1, to the culture. The cells were incubated for 4 days, washed with PBS, and treated with 1:5000 Versene for 10 minutes at 37° C. When necessary, the cells were detached by gentle cell scraping before being washed and analyzed. Each culture was resuspended in staining medium (Hanks's Balanced Salt Solution with 0.2% sodium bicarbonate, 0.01% sodium azide, 0.1 mM EDTA, 20 mM HEPES, and 2% fetal calf serum) and separated equally into six wells of a V bottom 96 well plate. The cells were pulse-spun at 2100 rpm on a Sorvall RTH-750 rotor, and resuspended in 25 μl of staining media. One microgram of specific phycoerythrin conjugated antibody was added to each well and incubated on ice for 45 minutes. The cells were washed three times, resuspended in 200 μl of 2% paraformaldehyde in PBS, and analyzed by flow cytometry with the Becton Dickinson FACScalibur. Gates were drawn on the forward v side scatter graph to remove contaminating cells from the analysis.

Results:

In this experiment, the differentiation of human monocytes to dendritic cells in response to IFN derived from the plasma of SLE patients blocked by treatment with 9D4-TM was measured by surface expression of two dendritic cell markers, CD38 and CD123. In FIG. 18. multiple serum samples from SLE patients failed to increase the surface expression of CD38 and CD123 in the presence of 9D4-TM. The IC50 values for 9D4-TM varied from 0.02 nM to 0.06 nM for both CD38 and CD123.

Conclusions:

The anti-IFNAR1 antibody 9D4-TM was able to block the ability of IFNα derived form SLE patients to induce pDC maturation as measured by cell surface marker expression.

6.19 Example 19: Anti-IFNAR Antibodies Suppress the Expression of CD38, CD123 and CD86 in Monocytes Stimulated with Leukocyte-IFN

Purpose:

In this example, the antibodies 9D4, 9D4-DM and 9D4 TM were tested for the inhibition of dendritic cell development, as assessed by the ability of the antibodies to inhibit the induction of the cell surface markers CD38, and CD123 by Leukocyte IFN.

Methods:

Monocytes were isolated from whole blood of healthy donors using a lymphocyte separation media (MP Biomedical, Solon Ohio) followed by positive selection using Monocyte Isolation kit II (Milteny Biotec, Auburn Calif.). Purified monocytes were then cultured at 1×10⁶ cells/mL in RPMI 1640 supplemented with 10% FBS (Gibco BRL). Serially diluted antibodies were prepared at final concentrations of 3 μg/mL-20 pg/mL in media and were added to appropriate wells of cells. After pre-incubation of approximately 5 minutes, 100 IU/mL of human leukocyte IFN (PBL Biomedical, Piscataway N.J.) was added to appropriate wells and cultures were incubated at 37° C., 5% CO₂ for 48 hours after which surface expression of CD38 and CD123 evaluated. Briefly, cells were pelleted at 1200 rpm for 5 minutes and culture media was removed from monolayers by aspiration followed by one wash 1× with sterile PBS. PBS was removed and 1 mL sterile cell dissociation buffer (Gibco BRL, Carlsbad Calif.) or 0.05% trypsin (Invitrogen, Carlsbad Calif.) was added to wells to remove cells from monolayers. After 5 minutes and brief agitation, equal volumes of RPMI 1640 supplemented with 10% FBS was added to each well, followed by two series of centrifugation and washes with sterile PBS. 50 μL of 1×PBS supplemented with 5% BSA (Sigma, St. Louis Mo.) and 10 μg/mL whole human IgG (Jackson ImmunoResearch Laboratories, West Grove Pa.) was added to each well for blocking of non-specific Fc antibody binding and incubated for 10 minutes at room temperature. 50 μL of 1×PBS supplemented with 5% BSA and PE-anti human CD123 and FITC-anti human CD38 antibodies (Becton Dickinson, Franklin Lakes N.J.) were added to appropriate wells and incubated for 30 minutes on ice. Cells were washed once in 1×PBS supplemented with 5% BSA and surface protein expression was measured on a BD LSRII (Becton Dickinson, Franklin Lakes N.J.).

Results:

Presented in FIG. 19 are the suppression curves of CD38 (A), CD123 (B), and CD86(C) expression exhibited by leukocyte-IFN stimulated PBMCs incubated with anti-IFNAR antibodies 9D4, 9D4DM, and 9D4TM. For each CD molecule, the anti-IFNAR antibodies elicited similar suppression curves which were utilized to generate IC50 values. For CD38 expression on PBMCs stimulated with leukocyte-IFN (A), the anti-IFNAR antibodies elicited IC50 values as follows: 9D4=4.3 ng/ml, 9D4DM=40 ng/ml, 9D4TM=25 ng/ml. For CD123 expression on PBMCs stimulated with leukocyte-IFN (B), the anti-IFNAR antibodies elicited IC50 values as follows: 9D4=7 ng/ml, 9D4DM=21 ng/ml, 9D4TM=10 ng/ml. For CD86 expression on PBMCs stimulated with leukocyte-IFN (C), the anti-IFNAR antibodies elicited IC50 values as follows: 9D4=20 ng/ml, 9D4DM=20 ng/ml, 9D4TM=26 ng/ml.

Conclusions:

The results in this Example demonstrate that antibodies of the invention, 9D4-DM and 9D4-TM exhibit similar suppression curves of IFN induction of pDC cell surface markers as compared to the parental 9D4 antibody.

6.20 Example 20: Modified Anti-IFNAR1 Antibodies Exhibit Decreased Binding to the Fc Receptor FcγRI

Purpose:

To demonstrate the reduced binding of a specific Fc receptor to modified anti-IFNAR1 antibodies.

Methods:

The binding activity of modified antibodies 9D4-DM and 9D4-TM to human FcγRI (CD64) was evaluated by ELISA. FcγRI in PBS (pH7.4) was coated at 25 μl/well in a microtiter plate (Costar cat. 3690) at the concentration of 20 μg/ml over night at 4° C. After washing and blocking with 4% milk 1 hr at room temperature, the biotinylated 9D4, 9D4TM, 9D4DM and control antibodies were added into the previously blocked plate and incubated at 37° C. for an hour, starting at 500 μg/ml and then in two fold serial dilution. The plate was washed with PBS (pH7.4) containing 0.05% of Tween 20 and 25 μl of HRP conjugated Avidin was added to each well. After an hour incubation at 37° C., the plates were washed again and 50 μl/well of substrate—SureBlue TMB peroxidase (KPL cat. 52-00-03) was added. The reaction was stopped with 50 μl of 0.2M H₂SO₄ after 5-10 minutes development. The ELISA signal was read at 450 nM.

Results:

In an ELISA based binding assay (FIG. 20), Modified anti-IFNAR1 antibodies 9D4DM and 9D4TM exhibited lower binding affinities to the FcγRI that the unmodified 9D4WT antibody as well as the control antibody.

Conclusions:

These results demonstrate that the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM elicit a lower affinity for the Fc receptor FcγRI as compared to the unmodified 9D4 antibody. The lowered affinity for the FcγRI receptor would lead to a lower induction of ADCC.

6.21 Example 21: The Fc Receptor FcγRIIIA Exhibits Reduced Binding to the Modified Anti-IFNAR1 Antibodies

Purpose:

To demonstrate the reduced binding of a specific Fc receptor to the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM as compared to the unmodified anti-IFNAR1 antibody 9D4.

Methods:

Fifty μg/ml of 9D4, 9D4TM, and 9D4DM antibodies diluted in PBS were coated on Immunlon IV microtiter plate over night at 4° C. After washing and blocking with 4% milk 1 hr at room temperature FcγRIIIA variants 158F (low affinity) and 158V (high affinity) with Flag tag were added to the wells of the blocked plate, starting at 50 μg/ml then in two-fold serial dilution. The plate was washed one hour later and incubated with biotin conjugated anti Flag antibody (Sigma) at 2 μg/ml. After washing 25 μl of HRP conjugated Avidin was added to each well. The unbound materials were removed by washing one hour after incubation. The binding signal was detected with the substrate TMB.

Results:

The results from an ELISA based binding assay between anti-IFNAR1 antibodies (9D4WT, 9D4DM, and 9D4TM) and the high and low affinity Fc receptor FcγRIIIA are presented in FIGS. 21(A, B, C). In FIG. 21(A) 9D4WT antibodies coated on the ELISA plate efficiently bind the high affinity FcγRIIIA receptor at concentrations greater than 3 ng/ml while there is limited binding of the low affinity FcγRIIIA receptor at all concentrations tested. In FIG. 21(B) Modified 9D4DM antibodies coated on the ELISA plate do not efficiently bind the high or low affinity FcγRIIIA receptors at any concentrations tested. Likewise, in FIG. 21(C) Modified 9D4TM antibodies coated on the ELISA plate do not efficiently bind the high or low affinity FcγRIIIA receptors at any concentrations tested.

Conclusions:

These results suggest that the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM have a decreased affinity for the FcγRIIIA receptor as compared to the unmodified anti-IFNAR1 antibody 9D4. Additionally, the decreased affinity for the specific Fc receptor could lead to a decrease in ADCC effector function.

6.22 Example 22: The Modified Antibodies 9D4DM and 9D4TM Exhibit Reduced Binding for the Fc Receptor FcγRIIIA

Purpose:

To demonstrate the reduced binding of a specific Fc receptor to modified antibodies 9D4DM and 9D4TM.

Methods:

Fifty g/ml of FcγRIIIA variants (FcγRIIIA-10 158F and FcγRIIIA-10 158V) in PBS were coated on Immunlon IV microtiter plate over night at 4° C. After washing and blocking with 4% milk 1 hr at room temperature biotinylated 9D4, 9D4TM, and 9D4DM antibodies were added to the wells of the blocked plate at 100 μg/mL. The plate was washed one hour later and incubated with HRP conjugated Avidin. The unbound materials were removed by washing one hr after incubation. The binding signal was detected with the substrate TMB.

Results:

The results from an ELISA based binding assay between the high and low affinity Fc receptors FcγRIIIA and anti-IFNAR1 antibodies (9D4WT, 9D4DM, and 9D4TM) are presented in FIGS. 22(A, B, C). In FIG. 22(A) the unmodified anti-IFNAR1 antibody 9D4, at concentrations greater than 3 ng/ml, efficiently binds the high affinity FcγRIIIA receptor immobilized on the ELISA plate, whereas the antibody demonstrates limited binding to the immobilized low affinity FcγRIIIA receptor at all concentrations tested. In FIG. 22(B) the modified anti-IFNAR1 antibody 9D4DM does not efficiently bind the immobilized high or low affinity FcγRIIIA receptors at any concentrations tested compared to the unmodified 9D4WT anti-IFNAR1 antibody. Likewise, in FIG. 22(C) the modified anti-IFNAR1 antibody 9D4TM does not efficiently bind the immobilized high or low affinity FcγRIIIA receptors at any concentrations tested compared to the unmodified 9D4WT anti-IFNAR1 antibody.

Conclusions:

This Example demonstrates that the modified antibodies 9D4DM and 9D4TM, exhibit decreased affinity for the Fc receptor, FcγRIIIA as compared to the parental unmodified 9D4 antibody. This reduced affinity could lead to a decrease in FcγRIIIA mediated ADCC effector function as compared to the parental antibody.

6.23 Example 23: Neutralization of IFNα Subtypes by Anti-IFNAR1 Antibodies

Purpose:

To demonstrate the ability of the anti-IFNAR1 antibodies MDX-1333, 9D4WT, and 9D4TM to neutralize specific IFNα subtypes in a reporter assay

Methods:

Reporter assays for IFNα neutralization have been well documented in the art. In this example, IFNα neutralization is measured by a HiL3 based reporter assay. An example of how a IFNα neutralization assay using HiL3 cells as a reporter is as follows: A human hepatoma cell line HiL3 was transfected with a plasmid containing an IFNα stimulated response element-luciferase (ISRE-Luc), and a neomycin resistance gene. These cells were kindly provided by Dr Michael Tovey (CNRS, Paris, France). Hil3, 30,000 cells/well, was cultured in white reflective 96 well plates (DYNEX Microlite) and grown overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1 mg/ml G418 (+penicillin/streptomycin/L-glutamine). After this incubation, various forms of interferon were added and the plates were cultured for 18 hours. The reaction was terminated by adding 10 ml of lysis buffer to luciferase substrate vial (Luc Lite Plus kit, Perkin-Elmer); 100 μl of this substrate solution was added to each well and read on Top Count for 10 minutes (10 minutes waiting in the dark, then 1 second read/well). The counts per second (cps) at each IFN concentration were determined and the IFN concentration or cps in each sample was calculated from the IFN titration curve using Prism software (San Diego, Calif.) with linear regression parameters.

Results:

The neutralization capacity for anti-IFNAR1 antibodies for various IFN species in a HiL3 reporter assay is presented in FIGS. 23 (A-E). The anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM inhibit multiple Type I interferon subtypes with similar potency. The anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFNα10 (A) with IC50 values of 0.09880 μg/ml, 0.008345 μg/ml, and 0.004287 μg/ml respectively. The anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize Human Leukocyte IFN (B) with IC50 values of 1.121 μg/ml, 0.02104 μg/ml, and 0.02120 μg/ml respectively. The anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFNα 2b (C) with IC50 values of 0.0006462 μg/ml, 0.002789 μg/ml, and 0.0008279 μg/ml respectively. The anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFNω (D) with IC50 values of 5.323 μg/ml, 0.01015 μg/ml, and 0.01423 μg/ml respectively. The anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFNβ (E) with IC50 values of 18.97 jag/ml, 0.7403 μg/ml, and 0.2611 μg/ml respectively.

Conclusions:

These results indicate that the anti-IFNAR1 antibodies MDX-1333, 9D4WT (unmodified) and 9D4TM (modified) exhibit similar neutralization specificity and capacity for multiple Type I interferons.

6.24 Example 24: Anti-IFNAR1 Antibodies Neutralize Type I IFN in Plasma from SLE Patients

Purpose:

To demonstrate the ability of anti-IFNAR1 antibodies to neutralize Type I IFN in plasma isolated from SLE patients as measured by a report assay.

Methods:

Stably transfected PIL-5 ISRE cells were maintained in RPMI 1640+1× Pen-strep-glutamine+10% FBS and seeded at 100,000 cells per well in Optilix white/clear 96 well plates (VWR, West Chester Pa.). Antibodies were titrated added to appropriate wells for a final concentration ranging from 90 μg/mL 60 pg/mL. Type-I interferon positive human SLE patient serum samples were added to each well for a final serum percentage of 50% per well. Cells, IFN, and antibodies were incubated overnight at 37° C., 5% CO₂. After overnight incubation, cells were pelleted briefly at 1200 rpm for 5 minutes and amplification of the luciferase protein was evaluated by lysing the cells with Cell Culture Lysis reagent and visualization using the Luciferase Assay System (Promega, Madison Wis.). Signal was measured in cps and IC50 curves were generated using GraphPad Prism 4 analysis software.

Results:

9D4-TM neutralizes Type I interferons in SLE patient plasma. The results from a neutralization assay of Type I interferons in SLE patient plasma is presented in FIG. 24. Neutralization of Type I interferon contained in the SLE patient plasma sample is specifically neutralized with 9D4-TM versus an Isotype control at increasing antibody concentrations. Specifically, 9D4-TM exhibits an IC50 of 0.04 nM for neutralization of Type I interferons in this plasma sample taken from an SLE patient.

Conclusions:

This result suggests that the modified anti-IFNAR1 antibody 9D4-TM has the capacity to effectively neutralize Type I interferon in SLE patients.

6.25 Example 25: Anti-IFNAR Antibodies Suppress the IFNα Induced pDC Population in PBMCs

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to suppress the accumulation of pDC cells in the peripheral blood of mice from a model of SLE.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. PBMCs were isolated from Spleen, Lymph Nodes, Bone Marrow and Peripheral Blood using standard isolation techniques and stained for the B220 and Ly6C surface markers. Isolated PBMCs were analyzed by FACS and double positive (B220 and Ly6C) cells were scored as pDC cells and the relative populations are represented in FIG. 25.

Results:

As represented in FIG. 25, ectopic expression of IFNα triggers an increase in pDC cells within the PBMCs isolated from spleen (A), lymph nodes (B), blood (C), and bone marrow (D) in the presence of PBS or mouse non-specific IgG. Mice treated with anti-IFNAR antibodies do not accumulate pDC cells in response to IFN-alpha. Mice treated with control Adenovirus do not accumulate pDCs in the PBMC population.

Conclusions:

These results suggest that anti-IFNAR antibodies specifically block the IFNα induced upregulation of pDC cells.

6.26 Example 26: Modified Anti-IFNAR1 Antibodies Exhibit Lower Binding Affinities to Fc Receptors

Purpose:

To evaluate the relative binding affinities of the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM with the parental unmodified antibody 9D4 to various Fc receptors.

Methods:

All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Uppsala, Sweden). In a typical experiment 1 μM solutions of 9D4 IgGs were used to immobilize anywhere from ˜7000 RUs-˜11,000 RUs of protein onto CM5 sensor chip surfaces using a standard amino coupling protocol (BIAcore, Inc.). Separately, a blank surface was also prepared on each chip using the identical protocol, minus the protein. This blank surface was used as a reference cell throughout the experiment, and served to correct for both non-specific binding and certain housekeeping artifacts. For the test-binding experiments, FcγRI was prepared at 20 nM in HBS-EP buffer (BIAcore, Inc., consisting of the following: 10 mM HEPES buffer, pH7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20. Between FcγRI injections, the IgG surface was regenerated with a 1 min. injection of 5 mM HCl. Sensorgram overlays were generated using the BIAevaluation 4.1 software (BIAcore, Inc, Uppsala, Sweden).

Results:

The anti-IFNAR1 antibody 9D4 and modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM were tested for binding affinity to immobilized FcγRI protein in a BIAcore assay format. As depicted in FIG. 26, the anti-IFNAR1 antibody 9D4 exhibits a high affinity for the immobilized FcγRI. The binding of the anti-IFNAR1 antibody 9D4 to FcγRI is specific as the similar assay run with ovalbumin exhibits very little affinity for the immobilized receptor. The modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM exhibit a lower affinity of the immobilized receptor FcγRI compared to the unmodified 9D4 anti-IFNAR1 antibody.

Conclusions:

The resultant lower affinities for FcγRI exhibited by the modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM suggest that these antibodies would have a diminished capacity to activate ADCC in vivo.

6.27 Example 27: Fc Receptors Exhibit Reduced Binding Affinities to Modified Anti-IFNAR1 Antibodies

Purpose:

To evaluate the relative binding affinities of various Fc receptors to the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM and the parental unmodified anti-IFNAR1 antibody 9D4.

Methods:

Surface Plasmon Resonance Measurements

All experiments were performed on a BIAcore 3000 instrument (BIAcore, Inc., Uppsala, Sweden). In a typical experiment a 1 μM solution of FcγRI was used to immobilize anywhere from ˜7000 RUs-˜11,000 RUs of protein onto CM5 sensor chip surfaces using a standard amino coupling protocol (BIAcore, Inc.). Separately, a blank surface was also prepared on each chip using the identical protocol, minus the protein. This blank surface was used as a reference cell throughout the experiment, and served to correct for both non-specific binding and certain housekeeping artifacts. For the test-binding experiments, antibodies were prepared at 333 nM in HBS-EP buffer (BIAcore, Inc., consisting of the following: 10 mM HEPES buffer, pH7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20. Between antibody injections, the FcγRI surface was regenerated with a 1 min. injection of 3M MgCl2. Sensorgram overlays were generated using the BIAevaluation 4.1 software (BIAcore, Inc, Uppsala, Sweden).

Results:

The anti-IFNAR1 antibodies 9D4, 9D4-TM and 9D4-DM were immobilized and incubated with soluble FcγRI. Binding affinity of the soluble FcγRI receptor to each of the anti-IFNAR1 antibodies were measured in a BIAcore assay and the resultant tracings are represented in FIGS. 27 A, B, C. The FcγRI bound the immobilized anti-IFNAR1 antibody 9D4 with a high affinity as represented in FIG. 27A. This interaction was highly specific as soluble ovalbumin did not show any binding to the immobilized anti-IFNAR1 antibody 9D4. The modified antibodies 9D4-TM and 9D4-DM do not bind the FcγRI as strongly as the wild type unmodified 9D4 antibody. In FIG. 27B, the modified anti-IFNAR1 antibody 9D4-DM was immobilized and incubated with either soluble FcγRI or ovalbumin. The FcγRI exhibited a low binding affinity for the immobilized 9D4-DM antibody. This binding affinity is similar to the non-specific interaction seen with soluble ovalbumin. In FIG. 27C, the modified anti-IFNAR1 antibody 9D4-TM was immobilized and incubated with either soluble FcγRI or ovalbumin. The FcγRI exhibited a low binding affinity for the immobilized 9D4-TM antibody. This binding affinity is similar to the non-specific interaction seen with soluble ovalbumin.

Conclusions:

The lower affinities exhibited by the Fc receptor FcγRI for the immobilized modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM over the unmodified anti-IFNAR1 antibody 9D4 suggests that the modified antibodies would exhibit a lower capacity to elicit an ADCC response.

6.28 Example 28: Anti-IFNAR Antibodies Block IFNα Responsive Gene Induction

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to block the induction of IFNα responsive genes in a mouse model of SLE.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. After 8 weeks into the experiment the mice were sacrificed and kidney tissue was removed. No more than 50 mg of tissue was used for RNA extraction using RLT lysis buffer (Qiagen). Samples were placed in lysis buffer and lysing matrix A (Qbiogene), and processed for 30 sec at 4.5 m/s using Fastprep24 homogenizer instrument (Thermo Electron Corporation, Waltham, Mass.). To isolate RNA, thawed tissue lysates were first processed using Qiashredder spin columns, then equal volumes of 70% ethanol were added to the tissue lysates and RNA was purified using Rneasy mini spin column kits according to the manufacturer's instruction. cDNA was generated from 3 μg of RNA using SuperScript III reverse transcriptase and oligo d(T) as described in the manufacturer's protocol (Invitrogen, Corp. Carlsbad, Calif.). Samples of cDNA were diluted in nuclease-free water and stored at −80° C.

Expression levels of selected genes were measured by real-time PCR TaqMan® analysis using the ABI 7900HT Fast Real-time PCR system (Applied Biosystems, Foster City, Calif.). Housekeeping gene β-actin was used for endogenous control. Reaction mixtures had a final volume of 20 μl consisting of 1 μl of cDNA, 2 μl of 20× primers and probes (TaqMan® Gene Expression Assays, Applied Biosystems) and 18 μl of diluted TaqMan® Fast Universal PCR Master Mix. Amplification conditions were: 20 seconds at 95° C., 50 cycles of 1 second at 95° C. and 20 seconds at 60° C. CT values range from 0 to 50, with the latter number assumed to represent no product formation. Quantification of gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to the housekeeping gene.

Results:

Presented in FIG. 28 are the results from a comparative expression analysis in the kidney of 6 genes induced by interferon alpha after 8 weeks in an accelerated lupus mouse model. Mice ectopically expressing interferon alpha were treated with mouse IgG or anti-IFNAR antibodies. After 8 weeks, the mice treated with control IgG demonstrated a high induction of IFNα responsive genes namely ICAM1, VCAM1, CXCL9, CXCL10, and IFIT1. Mice treated with anti-IFNAR antibodies did not show induction of IFNα responsive genes after 8 weeks.

Conclusions:

In the accelerated lupus mouse model treatment with anti-IFNAR antibodies blocks induction in the kidney of six genes (ICAM1, VCAM1, CXCL9, CXCL10, and IFIT1) mediated by the ectopically expression of IFN-alpha compared to control mice as measured by a Taqman assay. These results demonstrate that anti-IFNAR antibodies are capable of blocking IFNα mediated signaling in a SLE mouse model.

6.29 Example 29: Anti-IFNAR Antibodies Inhibit Accumulation of Autoantibodies in Serum

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to inhibit the accumulation of autoantibodies in serum of mice in an SLE model.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. Whole blood samples were taken at 1 week intervals from week 2-7 of the regimen. Serum anti-dsDNA autoantibody levels were assessed by ELISA. Briefly, ELISA plates pretreated with poly (L-lysine) (100 μg/ml) were coated with calf thymus activated DNA (5 μg/ml in carbonate-bicarbonate buffer) (SIGMA). After overnight incubation at 4° C., plates were blocked with PBS/10% FCS. Sera (1/200 dilution) were incubated for 30 minutes at room temperature. Bound IgG was detected with peroxidase-conjugated goat anti-mouse IgG (1/4000) (KPL) added to the plates for 30 min. Binding was measured by adding TMB substrate (KPL) and stop solution (KPL), and the OD was read at 450 nm. A mouse anti-ds DNA IgG standard in serum was run in serial dilution (from 625 ng/ml) (Alpha Diagnostic) on each plate to allow standardization.

Results:

Presented in FIG. 29 are the results from the ELISA based analysis of the levels of anti-ds DNA antibodies in mouse serum during an accelerated lupus mouse model time course. Mice ectopically expressing IFNα were treated with anti-IFNAR antibodies or mouse IgG control antibodies during an 7 week regimen. The mice treated with anti-IFNAR antibodies did not accumulate anti-dsDNA antibodies at the same rate or to the same extent of mice treated with control IgG antibodies. Mice infected with control adenovirus did not develop anti-ds DNA antibodies over the time course.

Conclusions:

These results demonstrate that anti-IFNAR antibodies reduced the accumulation of anti-dsDNA antibodies in response to elevated levels of IFN alpha.

6.30 Example 30: Anti-IFNAR Antibodies Reduce Proteinuria in the Accelerated Lupus Mouse Model

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to reduce established proteinuria (therapeutic setting) in the SLE mouse model.

Methods:

Mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. However, in a therapeutic approach, mice were allowed to develop proteinuria as a symptom of Lupus before application of the antibodies. Specifically, mice were allowed to develop a proteinuria score of 2.0-2.5 as described previously. Once the threshold level of proteinuria was passed, a treatment regimen of semi-weekly doses of PBS, control IgG or anti-IFNAR antibodies was conducted for 5 additional weeks. At semi-weekly intervals urine samples were tested and given a proteinuria score.

Results:

Presented in FIG. 30A are the results from a therapeutic study of anti-IFNAR antibodies reducing the proteinuria score of mice from an accelerated lupus model Briefly, mice were allowed to develop proteinuria at which time, the cohort was either given PBS, control IgG or anti-IFNAR antibodies as treatment. As documented within the figure, the proteinuria score decreased for only the group receiving anti-IFNAR antibodies. The mice receiving PBS or control IgG as treatment continued to increase the proteinuria score over time. (B) An analysis of the area under the curve for the results over the five weeks determined that the anti-IFNAR antibody treated group differed from the PBS alone or IgG control groups, both of which were very similar.

Conclusions:

These results demonstrate that anti-IFNAR antibodies could be used in a therapeutic setting of SLE.

6.31 Example 31: Anti-IFNAR Antibodies Reduce Mortality in the Accelerated Lupus Mouse Model

Purpose:

To demonstrate the ability of anti-IFNAR antibodies to reduce mortality in a therapeutic setting of the SLE lupus mouse model.

Methods:

Mice from the experimental procedures described in Example 30 also provided samples for analysis in this example. In a therapeutic approach, mice were allowed to develop proteinuria as a symptom of Lupus before application of the antibodies. Specifically, mice were allowed to develop a proteinuria score of 2.0-2.5 as described previously. Once the threshold level of proteinuria was passed, a treatment regimen of semi-weekly doses of PBS, control IgG or anti-IFNAR antibodies was conducted for 5 additional weeks. Overall mortality was tracked for an additional 4 weeks.

Results:

Presented in FIG. 31 are the mortality rates from a therapeutic study of anti-IFNAR antibodies in an accelerated lupus model Briefly, mice were allowed to develop proteinuria at which time, the cohort was either given PBS, control IgG or anti-IFNAR antibodies as treatment. Mice treated with anti-IFNAR antibodies exhibited no mortality at week 5, whereas mice treated with PBS or control IgG exhibited mortality rates of 87.5% and 62.5% respectively. Additionally, over the nine week study, anti-IFNAR treated animals exhibited a high survival rate compared to PBS or control IgG treated animals.

Conclusions:

The results in this Example demonstrate that anti-IFNAR antibodies can decrease the mortality associated with Lupus.

6.32 Example 32: Absence of 9D4-TM Mediated ADCC Activity

Purpose:

To verify that 9D4-TM is unable to induce ADCC activity, due to its poor binding affinity to FcγRI and FcγRIIIA a series of experiments were conducted.

Methods:

293F target cells were labeled with DiO cell label (Invitrogen, experiments I & II) and combined with unlabeled effectors PBMCs (for 4 h at 37° C., in the absence or presence 10 μg/ml of 9D4-TM, human IgG1 isotype negative control R3-47, 9D4-WT or anti-EphA2 antibody used as a positive control. Lysis of target cells was evaluated by measuring DiO⁺/PI⁺ (propidium iodide) double positive staining. Effector-target ratio=50-1, percent of lysis was calculated according to the formula: [(percent of double positive staining in the presence of antibodies−percent of double positive staining in media alone)/(percent of double positive staining in the presence of lysis buffer−percent of double positive staining in media alone)]. One hundred percent of lysis was achieved by adding lysis buffer (Promega).

Alternatively, 293F target cells were incubated with cells from a transgenic NK cell line stably expressing FcγRIIIA (experiment III) for 4 h at 37° C., in the absence or presence 10 μg/ml of 9D4-TM, human IgG1 isotype negative control R3-47, 9D4-wt or anti-EphA2 antibody used as a positive control. Effector-target ratio=4−1 and percent of lysis was calculated according to the formula: 100×(Experimental−Effector Spontaneous−Target Spontaneous)/(Target Maximum-Target Spontaneous).

On experiments I & II (PBMCs-293H ratio=50−1), percent of lysis was calculated according to the formula: [(percent of double positive staining in the presence of antibodies−percent of double positive staining in media alone)/(percent of double positive staining in the presence of lysis buffer−percent of double positive staining in media alone)]. On experiment III (Transgenic NK cell line expressing FcγIIIA-293H ratio=4−1), percent of lysis was calculated according to the formula: 100×(Experimental−Effector Spontaneous−Target Spontaneous)/(Target Maximum-Target Spontaneous).

Results:

The modified antibody 9D4-TM or the unmodified antibody 9D4-WT exhibited no detectable ADCC activity on 293F cells over that observed with the R3-47 antibody, (Table 4). In contrast, the positive control antibody, an anti-EphA2 antibody, caused a two-fold increase in cytotoxicity over background level. These results confirm that 9D4-TM cannot mediate ADCC on IFNAR1 expressing targets.

TABLE 5 Evaluation of ADCC activity of Anti-IFNAR1 antibodies. Exp. I Exp. II Exp. III Antibodies % of target lysis % of target lysis % of target lysis Positive control: 33 ± 4 36 ± 1 43.4 ± 0.5 Anti-EphA2 Negative control: 14 ± 1 18 ± 3 18.1 ± 1.1 R3-47 9D4-WT 14 ± 2 20 ± 2 17.5 ± 1.6 9D-TM 14 ± 2 20 ± 2 ND Exp. I/II/III: experiments I/II/III. ND: not done.

Conclusions:

These results demonstrate that modified anti-IFNAR1 antibody 9D4-TM does not stimulate detectable ADCC activity directed at IFNAR1 expressing target cells.

6.33 Example 33: Three-Dimensional Structures of Human Fc Region Comprising L234F/L235E/P331S Mutations

Purpose:

To determine the three-dimensional structures of human IgG1 Fc region comprising L234F/L235E/P331S mutations (Fc-TM).

Methods:

Purification of Fc-TM:

The human Fc/TM fragment was obtained from the enzymatic cleavage of 9D4-TM. Digestion was carried out using immobilized ficin according to the manufacturer's instructions (Pierce). Purification was first performed on HiTrap Protein A columns according to the manufacturer's instructions (GE Healthcare, Piscataway, N.J.). After dialysis in 50 mM NaOAc/pH 5.2, the protein solution was applied to a HiTrap SP HP column (GE Healthcare) and collected in the flow through. The flow through was loaded onto a HiTrap Q column (GE Healthcare) and eluted in a NaCl gradient to yield a homogenous Fc/TM preparation, as judged by reducing and non-reducing SDS-PAGE. Fc-TM SDS-PAGE profile showed the presence of only one band around 25 kDa or 50 kDa under reducing or non reducing conditions, respectively. This observation clearly demonstrated the presence of at least one interchain disulfide bond at positions C226 and/or C229. Consequently, mutated ‘downstream’ residues F234 and E235 were present in the polypeptide chain comprising the crystal.

Crystallization of Fc-TM:

Purified Fc-TM was concentrated to about 5 mg/ml using a Centricon concentrator (Millipore, Billerica Mass., 30 KDa cutoff). Crystallization conditions were identified using the commercial screens from Hampton Research (Hampton Research, Aliso Viejo, Calif.), Emerald BioSystems (Emerald BioSystems, Inc., Bainbridge Island, Wash.) and Molecular Dimensions (Molecular Dimensions Inc., Apopka, Fla.). Each screen yielded several potentially usable crystallization conditions. Upon optimization, diffraction-quality crystals were obtained from 0.2M Zinc acetate, 0.1M Imidazole-Malate, pH 8.0, 5% PEG 3350, 5% glycerol at protein concentration of 2.0 mg/ml. Under these conditions, well-shaped crystals with three dimensions ranging from 0.1 to 0.2 mm grew in 2-3 days.

Data Collection:

Diffraction data were collected from a single crystal at the Center for Advanced Research in Biotechnology (CARB, University of Maryland Biotechnology Institute, Rockville, Md.) using a Rigaku MicroMax™ 007 rotating anode generator with an R-AXIS IV++ imaging plate (Rigaku/MSC, The Woodlands, Tex.). Prior to cooling, the crystal was kept for a few minutes in its growth solution supplemented with 20% glycerol. The crystal was then cooled to 105 kelvins with an X-stream 2000 Cryogenic cooler (Rigaku/MSC). Diffraction of up to 2.3 Å was achieved after one round of annealing as described (Oganesyan et al., 2007). Diffraction data comprising 234 images were collected using an oscillation range of 0.5°, a crystal/detector distance of 200 mm and an exposure time of 600 s. Data were integrated and scaled using the HKL 2000 software (Otwinowski & Minor, 1997).

Structure Determination:

Molecular replacement, refinement, and electron density calculation were carried out using the CCP4 (Collaborative Computational Project) program suite. The C-face centered orthorhombic crystal had a 58% solvent content and V_(M) of 2.9, assuming one Fc polypeptide in the asymmetric unit of the cell. The crystal structure of Fc/TM was determined by molecular replacement and refined at 2.3 Å resolution. The human Fc structure corresponding to PDB ID number 2DTQ (Matsumiya et al., (2007) J. Mol. Biol. 368:767-779) was used as the model because of its high resolution and unliganded state. In particular, the C_(H)2 and C_(H)3 domains were considered separately to minimize any bias in terms of the domains relative conformation. Data up to 3.0 Å were used for the molecular replacement problem using Phaser (McCoy et al., (2005) Acta Cryst. D61, 458-464). After refinement of the solutions, the final LL-gain and the Z-score were 1192 and 31, respectively. Weighted electron density calculated with FWT/PHWT at 3.0 Å showed a good match with the model with the exception of some loops in the C_(H)2 and C_(H)3 domains. Strong positive difference electron density calculated with DELFWT/PHDELWT was visible in the expected place of N-linked carbohydrate residues attached to N297. There was no density present for any hinge residue preceding that at position 236, a result presumably attributable to the high flexibility of this region. It is noted that only two previously described unliganded human Fc structures could reveal positions 234 and 235 (2DTQ/2DTS; Matsumiya et al., (2007) J. Mol. Biol. 368:767-779). Likewise, residues at positions 446 and 447 could not be visualized. The residue at position 331 was first modeled as an alanine.

Several alternating rounds of refinement with ‘Refmac 5’ (Murshudov et al., (1997) Acta Cryst. D53, 240-255) and manual building using the “0” graphics software (Jones et al., (1991) Acta Cryst. A47, 110-119) converged with R_(factor) of 21.6 and Free R_(factor) of 27.5 for data up to 2.3 Å resolution. After the first round of refinement, the electron density allowed placement of the carbohydrates as well as substitution by a serine residue at position 331. At later stages of refinement, the model was analyzed using the TLS Motion Determination (TLSMD) program running on its web Server (Painter et al. (2006). J. Appl. Cryst. 39, 109-111; Painter et al. (2006) Acta Cryst. D62, 439-450). Further refinement was then carried out with Refmac 5 in TLS and restrained refinement mode using five distinct groups of residues (236-324, 325-341, 342-358, 359-403 and 404-445). Zinc ions present in the crystallization buffer were detected in the electron density and modeled as such when the coordination sphere and distance permitted. In particular, one zinc ion was found coordinated by H310 and H435. Another was coordinated by H285 and H268 of the symmetry related polypeptide. Two others were bound to E318 and E345. In all cases, water molecules completed the expected tetrahedral coordination sphere of the zinc ions. The carbohydrate moiety was modeled according to its electron density and the final model contained nine sugar residues, essentially as described by us in the context of another human Fc structure (Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, in press). The final model contained 75 solvent molecules. Crystallographic data and refinement statistics are given in Table 6.

TABLE 6 X-Ray data collection and model refinement statistics. Wavelength, {acute over (Å)} 1.54 Resolution, {acute over (Å)} 36.83 − 2.30 (2.38-2.30) ^(a) Space group C222₁ Cell parameters, {acute over (Å)} 50.18, 147.30, 75.47 Total reflections 54,409 Unique reflections 12,617 Average redundancy 4.31 (2.72) ^(a) Completeness, % 98.3 (90.0) ^(a) R_(merge) 0.062 (0.300) ^(a) I/σ(I) 13.0 (3.3) ^(a) R factor/Free R factor 0.216/0.275 RMSD bonds, {acute over (Å)} 0.012 RMSD angles, ° 1.48 Residues in most favored region 89.9 of { 

, ψ} space, % Residues in additionally 10.1 allowed region of { 

, ψ} space, % Number of protein atoms 1678 Number of non-protein atoms 189 B factor (Model/Wilson), {acute over (Å)}² 43/40 ^(a) Values in parentheses correspond to the highest resolution shell

Results:

Fc-TM crystallized in space group C222₁ with one polypeptide in the asymmetric unit (FIG. 32). The crystal diffracted to 2.3 Å resolution, and exhibited a relatively high average mosaicity of 1.26°. This high mosaicity appeared to be a property of both cooled and non-cooled crystals. All residues at positions 236 to 445 could be traced in the electron density and no electron density was observed for hinge residues prior to position 236, thus rendering the L234F and L235E mutations invisible. The electron density at position 331 corresponded to serine.

The atomic coordinates and experimental structure factors of Fc-TM have been deposited with the Protein Data Bank under accession number 3C2S.

The overall three-dimensional structure of Fc-TM was very similar to previously reported structures of unliganded human Fc regions (Deisenhofer, (1981). Biochemistry, 20: 2361-2370; Krapp et al., (2003). J. Mol. Biol. 325, 979-989; Matsumiya et al., (2007). J. Mol. Biol. 368:767-779; Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, in press). More precisely, the human Fc structures corresponding to PDB ID numbers 1H3W (Krapp et al., (2003). J. Mol. Biol. 325:979-989) and 2QL1 (Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, In the press) were closest to Fc-TM in terms of cell parameters, asymmetric unit content, space group and packing. When considered individually, Fc-TM C_(H)2 and C_(H)3 domains showed great structural conservation and rigidity when compared with other unliganded, unmutated human Fc structures. For instance, rms coordinate displacements of Cα atoms were 0.6 and 0.4 Å for the C_(H)2 and C_(H)3 domains, respectively, when superimposing Fc-TM with chain A of PDB ID number 2DTQ (Matsumiya et al., (2007). J. Mol. Biol. 368, 767-779).

Table 7 following below, provides the atomic structure coordinates of Fc-TM. The following abbreviations are used in Table 7

“Atom Type” refers to the element whose coordinates are provided. The first letter in the column defines the element.

“A.A.” refers to amino acid.

“X, Y and Z” provide the Cartesian coordinates of the element.

“B” is a thermal factor that measures movement of the atom around its atomic center.

“OCC” refers to occupancy, and represents the percentage of time the atom type occupies the particular coordinate. OCC values range from 0 to 1, with 1 being 100%.

TABLE 7 The atomic structure coordinates of Fc-TM REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM: REFMAC 5.2.0019 REMARK 3 AUTHORS: MURSHUDOV, VAGIN, DODSON REMARK 3 REMARK 3 REFINEMENT TARGET: MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS):   2.30 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS):   30.00 REMARK 3 DATA CUTOFF (SIGMA (F)): NONE REMARK 3 COMPLETENESS FOR RANGE (%):   98.43 REMARK 3 NUMBER OF REFLECTIONS: 11994 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING + TEST SET):  0.21928 REMARK 3 R VALUE (WORKING SET)  0.21637 REMARK 3 FREE R VALUE:  0.27541 REMARK 3 FREE R VALUE TEST SET SIZE (%):  4.9 REMARK 3 FREE R VALUE TEST SET COUNT: 619 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED:  20 REMARK 3 BIN RESOLUTION RANGE HIGH:  2.300 REMARK 3 BIN RESOLUTION RANGE LOW:  2.360 REMARK 3 REFLECTION IN BIN (WORKING SET): 794 REMARK 3 BIN COMPLETENESS (WORKING + TEST) (%):  89.74 REMARK 3 BIN R VALUE (WORKING SET):  0.242 REMARK 3 BIN FREE R VALUE SET COUNT:  46 REMARK 3 BIN FREE R VALUE:  0.342 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 ALL ATOMS: 1867 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2): NULL REMARK 3 MEAN B VALUE (OVERALL, A**2): 43.320 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2): −3.83 REMARK 3 B22 (A**2):  0.96 REMARK 3 B33 (A**2):  2.88 REMARK 3 B12 (A**2):  0.00 REMARK 3 B13 (A**2):  0.00 REMARK 3 B23 (A**2):  0.00 REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3 ESU BASED ON R VALUE (A):  0.327 REMARK 3 ESU BASED ON FREE R VALUE (A):  0.256 REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A):  0.194 REMARK 3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 14.024 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENT FO-FC: 0.941 REMARK 3 CORRELATION COEFFICIENT FO-FC FREE: 0.898 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK 3 BOND LENGTHS REFINED ATOMS (A): 1845 0.012 0.022 REMARK 3 BOND ANGLES REFINED ATOMS (DEGREES): 2527 1.482 2.032 REMARK 3 TORSION ANGLES, PERIOD 1 (DEGREES):  209 6.172 5.000 REMARK 3 TORSION ANGLES, PERIOD 2 (DEGREES):  76 33.844  25.000  REMARK 3 TORSION ANGLES, PERIOD 3 (DEGREES):  295 17.124  15.000  REMARK 3 TORSION ANGLES, PERIOD 4 (DEGREES):   6 20.037  15.000  REMARK 3 CHIRAL-CENTER RESTRAINTS (A**3):  302 0.085 0.200 REMARK 3 GENERAL PLANES REFINED ATOMS (A): 1323 0.005 0.020 REMARK 3 NON-BONDED CONTACTS REFINED ATOMS (A):  714 0.202 0.200 REMARK 3 NON-BONDED TORSION REFINED ATOMS (A): 1211 0.311 0.200 REMARK 3 H-BOND (X . . . Y) REFINED ATOMS (A):  85 0.168 0.200 REMARK 3 POTENTIAL METAL-ION REFINED ATOMS (A):   1 0.013 0.200 REMARK 3 SYMMETRY VDW REFINED ATOMS (A):  45 0.267 0.200 REMARK 3 SYMMETRY H-BOND REFINED ATOMS (A):  10 0.166 0.200 REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A**2): 1090 0.502 1.500 REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 1737 0.773 2.000 REMARK 3 SIDE-CHAIN BOND REFINED ATOMS (A**2):  850 1.312 3.000 REMARK 3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2):  790 2.117 4.500 REMARK 3 REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3 NUMBER OF NCS GROUPS: NULL REMARK 3 REMARK 3 REMARK 3 TLS DETAILS REMARK 3 NUMBER OF TLS GROUPS: 5 REMARK 3 ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY REMARK 3 REMARK 3 TLS GROUP: 1 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: A 236  A 324 REMARK 3 ORIGIN FOR THE GROUP (A): 8.3389  24.1913  −4.5478 REMARK 3 T TENSOR REMARK 3 T11:  0.0215 T22:  0.0920 REMARK 3 T33:  0.3541 T12:  0.0433 REMARK 3 T13: −0.0938 T23: −0.3463 REMARK 3 L TENSOR REMARK 3 L11:  5.5174 L22:  6.9851 REMARK 3 L33:  1.3110 L12:  0.6985 REMARK 3 L13: −0.3877 L23:  1.4474 REMARK 3 S TENSOR REMARK 3 S11:  0.0024 S12: −0.9714 S13:  1.6061 REMARK 3 S21:  0.4006 S22:  0.0112 S23: −0.5043 REMARK 3 S31: −0.2230 S32: −0.0083 S33: −0.0136 REMARK 3 REMARK 3 TLS GROUP: 2 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: A 325  A 341 REMARK 3 ORIGIN FOR THE GROUP (A): 6.2355  28.7737  −13.4151 REMARK 3 T TENSOR REMARK 3 T11:  0.4194 T22:  0.0438 REMARK 3 T33:  0.6367 T12:  0.0309 REMARK 3 T13: −0.1209 T23: −0.1743 REMARK 3 L TENSOR REMARK 3 L11:  2.0696 L22:  7.3867 REMARK 3 L33:  3.9900 L12:  0.5828 REMARK 3 L13: −0.3193 L23:  2.0049 REMARK 3 S TENSOR REMARK 3 S11: −0.3128 S12: −0.3347 S13:  1.6116 REMARK 3 S21: −0.6048 S22:  0.4400 S23:  0.4114 REMARK 3 S31: −1.6055 S32:  0.0271 S33: −0.1271 REMARK 3 REMARK 3 TLS GROUP: 3 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: A 342  A 358 REMARK 3 ORIGIN FOR THE GROUP (A): 19.6741  −9.9102  −17.8082 REMARK 3 T TENSOR REMARK 3 T11:  0.0147 T22: −0.0558 REMARK 3 T33:  0.2412 T12:  0.0130 REMARK 3 T13: −0.0465 T23:  0.0419 REMARK 3 L TENSOR REMARK 3 L11:  5.9397 L22:  3.4770 REMARK 3 L33:  1.3027 L12: −0.2675 REMARK 3 L13: −2.7731 L23:  0.2922 REMARK 3 S TENSOR REMARK 3 S11:  0.1902 S12:  0.1053 S13: −2.1005 REMARK 3 S21: −0.2927 S22: −0.5125 S23: −0.3505 REMARK 3 S31:  0.2359 S32: −0.0277 S33:  0.3223 REMARK 3 REMARK 3 TLS GROUP: 4 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: A 359  A 403 REMARK 3 ORIGIN FOR THE GROUP (A): 21.2651  −3.5914  −12.2859 REMARK 3 T TENSOR REMARK 3 T11: −0.1689 T22: −0.0639 REMARK 3 T33: −0.1638 T12:  0.0043 REMARK 3 T13:  0.0241 T23:  0.0801 REMARK 3 L TENSOR REMARK 3 L11: 12.4510 L22:  2.7911 REMARK 3 L33:  2.9332 L12:  0.0470 REMARK 3 L13:  0.1119 L23: −0.2768 REMARK 3 S TENSOR REMARK 3 S11: −0.1346 S12: −1.2217 S13: −1.1281 REMARK 3 S21:  0.1580 S22:  0.0409 S23: −0.1830 REMARK 3 S31:  0.0059 S32:  0.2154 S33:  0.0937 REMARK 3 REMARK 3 TLS GROUP: 5 REMARK 3 NUMBER OF COMPONENTS GROUP: 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE: A 404  A 445 REMARK 3 ORIGIN FOR THE GROUP (A): 19.4718  −9.7512  −9.1313 REMARK 3 T TENSOR REMARK 3 T11: −0.0158 T22:  0.1994 REMARK 3 T33:  0.1938 T12:  0.0293 REMARK 3 T13:  0.0582 T23:  0.3819 REMARK 3 L TENSOR REMARK 3 L11: 13.1107 L22:  0.0678 REMARK 3 L33:  1.6932 L12:  0.9209 REMARK 3 L13: −1.5605 L23: −0.0412 REMARK 3 S TENSOR REMARK 3 S11: −0.1532 S12: −2.3239 S13: −2.6014 REMARK 3 S21: −0.0410 S22: −0.1484 S23: −0.1293 REMARK 3 S31:  0.3788 S32:  0.2592 S33:  0.3017 REMARK 3 REMARK 3 REMARK 3 BULK SOLVENT MODELLING. REMARK 3 METHOD USED: MASK REMARK 3 PARAMETERS FOR MASK CALCULATION REMARK 3 VDW PROBE RADIUS: 1.20 REMARK 3 ION PROBE RADIUS: 0.80 REMARK 3 SHRINKAGE RADIUS: 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: NULL REMARK 3 SSBOND 1 CYS A  321  CYS A  261 SSBOND 2 CYS A  425  CYS A  367 LINK C1 NAG C 1 1.439 ND2 ASN A 297 NAG-ASN CISPEP 1 TYR A  373  PRO A  374 0.00 LINK NAG C 1 NAG C 2 BETA1-4 LINK NAG C 2 BMA C 3 BETA1-4 LINK BMA C 3 MAN C 4 ALPHA1-3 LINK MAN C 4 NAG C 5 BETA1-2 LINK BMA C 3 MAN C 7 ALPHA1-6 LINK MAN C 7 NAG C 8 BETA1-2 LINK NAG C 8 GAL C 9 BETA1-4 LINK NAG C 1 FUC C 11 ALPHA1-6 MODRES NAG C 1 NAG-b-D RENAME MODRES NAG C 2 NAG-b-D RENAME MODRES MAN C 4 MAN-a-D RENAME MODRES NAG C 5 NAG-b-D RENAME MODRES MAN C 7 MAN-a-D RENAME MODRES NAG C 8 NAG-b-D RENAME MODRES GAL C 9 GAL-b-D RENAME MODRES FUC C 11  FUC-a-L RENAME CRYST1 50.178  147.301  75.473  90.00  90.00  90.00 C 2  2 21 SCALE1 0.019929 0.000000 0.000000 0.00000 SCALE2 0.000000 0.006789 0.000000 0.00000 SCALE3 0.000000 0.000000 0.013250 0.00000 ATOM 1 N GLY A 236 18.122 39.286 −14.907 1.00 50.67 N ANISOU 1 N GLY A 236 6366 6478 6407 30 −8 −27 N ATOM 2 CA GLY A 236 17.938 40.336 −13.862 1.00 50.37 C ANISOU 2 CA GLY A 236 6370 6447 6319 23 15 16 C ATOM 3 C GLY A 236 17.092 39.872 −12.683 1.00 50.35 C ANISOU 3 C GLY A 236 6337 6451 6340 0 7 36 C ATOM 4 O GLY A 236 17.603 39.755 −11.559 1.00 50.77 O ANISOU 4 O GLY A 236 6425 6518 6346 −19 −27 64 O ATOM 5 N GLY A 237 15.805 39.607 −12.942 1.00 49.94 N ANISOU 5 N GLY A 237 6294 6360 6321 −7 22 32 N ATOM 6 CA GLY A 237 14.821 39.264 −11.889 1.00 48.94 C ANISOU 6 CA GLY A 237 6194 6188 6211 20 42 32 C ATOM 7 C GLY A 237 15.074 37.906 −11.254 1.00 48.37 C ANISOU 7 C GLY A 237 6128 6107 6142 17 76 5 C ATOM 8 O GLY A 237 16.078 37.256 −11.568 1.00 48.88 O ANISOU 8 O GLY A 237 6209 6156 6205 47 90 −11 O ATOM 9 N PRO A 238 14.186 37.462 −10.336 1.00 47.63 N ANISOU 9 N PRO A 238 6027 5985 6082 15 57 −17 N ATOM 10 CA PRO A 238 14.432 36.144 −9.746 1.00 46.76 C ANISOU 10 CA PRO A 238 5926 5876 5964 31 25 −22 C ATOM 11 CB PRO A 238 13.327 36.008 −8.686 1.00 46.65 C ANISOU 11 CB PRO A 238 5911 5868 5945 11 −23 3 C ATOM 12 CG PRO A 238 12.878 37.422 −8.404 1.00 46.85 C ANISOU 12 CG PRO A 238 5930 5861 6007 30 38 0 C ATOM 13 CD PRO A 238 12.974 38.083 −9.771 1.00 47.56 C ANISOU 13 CD PRO A 238 6038 5947 6084 21 63 −44 C ATOM 14 C PRO A 238 14.308 35.056 −10.800 1.00 46.45 C ANISOU 14 C PRO A 238 5899 5823 5925 31 3 −16 C ATOM 15 O PRO A 238 13.803 35.311 −11.898 1.00 46.74 O ANISOU 15 O PRO A 238 5942 5884 5930 50 −21 −25 O ATOM 16 N SER A 239 14.806 33.868 −10.471 1.00 46.09 N ANISOU 16 N SER A 239 5876 5767 5868 24 12 −30 N ATOM 17 CA SER A 239 14.710 32.689 −11.333 1.00 45.35 C ANISOU 17 CA SER A 239 5783 5670 5778 −8 43 −6 C ATOM 18 CB SER A 239 16.093 32.273 −11.833 1.00 45.44 C ANISOU 18 CB SER A 239 5822 5669 5773 12 73 −51 C ATOM 19 OG SER A 239 16.516 33.126 −12.892 1.00 46.53 O ANISOU 19 OG SER A 239 6055 5754 5871 22 122 1 O ATOM 20 C SER A 239 14.112 31.580 −10.496 1.00 44.68 C ANISOU 20 C SER A 239 5695 5601 5679 8 13 0 C ATOM 21 O SER A 239 14.492 31.423 −9.338 1.00 44.81 O ANISOU 21 O SER A 239 5689 5655 5681 51 18 72 O ATOM 22 N VAL A 240 13.161 30.845 −11.077 1.00 43.99 N ANISOU 22 N VAL A 240 5587 5491 5634 −1 45 2 N ATOM 23 CA VAL A 240 12.474 29.760 −10.386 1.00 43.00 C ANISOU 23 CA VAL A 240 5453 5401 5482 −1 37 −22 C ATOM 24 CB VAL A 240 10.932 29.909 −10.474 1.00 43.13 C ANISOU 24 CB VAL A 240 5481 5408 5496 8 59 −20 C ATOM 25 CG1 VAL A 240 10.217 28.789 −9.696 1.00 42.84 C ANISOU 25 CG1 VAL A 240 5433 5309 5533 54 −34 −15 C ATOM 26 CG2 VAL A 240 10.519 31.239 −9.927 1.00 43.46 C ANISOU 26 CG2 VAL A 240 5572 5367 5572 −2 12 0 C ATOM 27 C VAL A 240 12.868 28.427 −10.986 1.00 42.45 C ANISOU 27 C VAL A 240 5377 5353 5396 −10 11 −1 C ATOM 28 O VAL A 240 12.936 28.272 −12.207 1.00 42.69 O ANISOU 28 O VAL A 240 5376 5426 5419 −44 62 −24 O ATOM 29 N PHE A 241 13.128 27.468 −10.108 1.00 41.87 N ANISOU 29 N PHE A 241 5300 5311 5296 −10 1 −22 N ATOM 30 CA PHE A 241 13.405 26.097 −10.498 1.00 40.91 C ANISOU 30 CA PHE A 241 5188 5170 5185 −31 −20 −52 C ATOM 31 CB PHE A 241 14.884 25.757 −10.294 1.00 41.14 C ANISOU 31 CB PHE A 241 5199 5228 5204 −15 −44 −34 C ATOM 32 CG PHE A 241 15.799 26.534 −11.203 1.00 41.60 C ANISOU 32 CG PHE A 241 5289 5240 5274 −81 3 −5 C ATOM 33 CD1 PHE A 241 16.448 27.682 −10.744 1.00 41.55 C ANISOU 33 CD1 PHE A 241 5248 5213 5326 −52 1 −15 C ATOM 34 CE1 PHE A 241 17.271 28.424 −11.601 1.00 40.85 C ANISOU 34 CE1 PHE A 241 5052 5159 5308 −21 −14 −18 C ATOM 35 CZ PHE A 241 17.448 28.019 −12.917 1.00 40.46 C ANISOU 35 CZ PHE A 241 5124 5094 5154 −75 22 −43 C ATOM 36 CE2 PHE A 241 16.792 26.872 −13.391 1.00 42.17 C ANISOU 36 CE2 PHE A 241 5259 5419 5342 51 84 −27 C ATOM 37 CD2 PHE A 241 15.978 26.143 −12.537 1.00 41.34 C ANISOU 37 CD2 PHE A 241 5253 5148 5303 −103 60 −23 C ATOM 38 C PHE A 241 12.493 25.189 −9.716 1.00 40.07 C ANISOU 38 C PHE A 241 5090 5072 5061 −21 −17 −53 C ATOM 39 O PHE A 241 12.175 25.475 −8.572 1.00 40.18 O ANISOU 39 O PHE A 241 5110 5045 5111 −37 −101 −113 O ATOM 40 N LEU A 242 12.044 24.109 −10.356 1.00 39.75 N ANISOU 40 N LEU A 242 5035 5028 5040 6 13 −59 N ATOM 41 CA LEU A 242 11.017 23.235 −9.794 1.00 38.92 C ANISOU 41 CA LEU A 242 4944 4899 4942 2 21 −61 C ATOM 42 CB LEU A 242 9.663 23.546 −10.438 1.00 38.70 C ANISOU 42 CB LEU A 242 4941 4864 4898 −18 4 −84 C ATOM 43 CG LEU A 242 8.396 22.862 −9.936 1.00 38.46 C ANISOU 43 CG LEU A 242 4855 4805 4951 71 −15 −74 C ATOM 44 CD1 LEU A 242 8.085 23.234 −8.504 1.00 37.91 C ANISOU 44 CD1 LEU A 242 4727 4827 4849 50 −49 −62 C ATOM 45 CD2 LEU A 242 7.275 23.271 −10.825 1.00 38.22 C ANISOU 45 CD2 LEU A 242 4846 4868 4806 137 −149 −134 C ATOM 46 C LEU A 242 11.409 21.793 −10.021 1.00 39.20 C ANISOU 46 C LEU A 242 4972 4961 4959 −11 13 −31 C ATOM 47 O LEU A 242 11.605 21.361 −11.149 1.00 39.40 O ANISOU 47 O LEU A 242 5021 4924 5024 −24 63 −55 O ATOM 48 N PHE A 243 11.510 21.044 −8.936 1.00 39.65 N ANISOU 48 N PHE A 243 4992 5044 5028 12 −12 −43 N ATOM 49 CA PHE A 243 12.230 19.792 −8.968 1.00 39.97 C ANISOU 49 CA PHE A 243 5044 5044 5099 −24 −2 −51 C ATOM 50 CB PHE A 243 13.404 19.823 −7.970 1.00 40.13 C ANISOU 50 CB PHE A 243 5022 5064 5162 −8 40 −26 C ATOM 51 CG PHE A 243 14.424 20.876 −8.279 1.00 41.34 C ANISOU 51 CG PHE A 243 5170 5154 5381 −72 6 −95 C ATOM 52 CD1 PHE A 243 14.319 22.156 −7.719 1.00 42.25 C ANISOU 52 CD1 PHE A 243 5360 5249 5441 34 −36 −90 C ATOM 53 CE1 PHE A 243 15.269 23.146 −8.016 1.00 41.11 C ANISOU 53 CE1 PHE A 243 5158 5065 5394 −69 72 −88 C ATOM 54 CZ PHE A 243 16.321 22.865 −8.888 1.00 41.69 C ANISOU 54 CZ PHE A 243 5217 5205 5418 −88 −1 −97 C ATOM 55 CE2 PHE A 243 16.435 21.587 −9.458 1.00 42.21 C ANISOU 55 CE2 PHE A 243 5279 5206 5551 −93 135 −80 C ATOM 56 CD2 PHE A 243 15.487 20.603 −9.154 1.00 42.21 C ANISOU 56 CD2 PHE A 243 5308 5220 5508 −31 66 −136 C ATOM 57 C PHE A 243 11.275 18.673 −8.648 1.00 40.06 C ANISOU 57 C PHE A 243 5009 5104 5107 −37 −10 −64 C ATOM 58 O PHE A 243 10.462 18.809 −7.729 1.00 39.94 O ANISOU 58 O PHE A 243 4936 5131 5107 −18 −13 −92 O ATOM 59 N PRO A 244 11.369 17.561 −9.405 1.00 40.05 N ANISOU 59 N PRO A 244 5036 5044 5138 −26 19 −78 N ATOM 60 CA PRO A 244 10.474 16.436 −9.204 1.00 39.94 C ANISOU 60 CA PRO A 244 5074 5000 5099 −2 16 −66 C ATOM 61 CB PRO A 244 10.819 15.510 −10.373 1.00 40.24 C ANISOU 61 CB PRO A 244 5091 5057 5139 −22 19 −62 C ATOM 62 CG PRO A 244 12.252 15.834 −10.700 1.00 40.53 C ANISOU 62 CG PRO A 244 5047 5064 5287 −14 −26 −70 C ATOM 63 CD PRO A 244 12.334 17.315 −10.494 1.00 40.31 C ANISOU 63 CD PRO A 244 5051 5061 5203 −28 −12 −57 C ATOM 64 C PRO A 244 10.810 15.760 −7.881 1.00 39.34 C ANISOU 64 C PRO A 244 5004 4929 5012 10 −25 −82 C ATOM 65 O PRO A 244 11.848 16.049 −7.315 1.00 39.44 O ANISOU 65 O PRO A 244 5071 4916 4996 11 −14 −147 O ATOM 66 N PRO A 245 9.943 14.861 −7.397 1.00 38.79 N ANISOU 66 N PRO A 245 4967 4861 4907 1 −10 −50 N ATOM 67 CA PRO A 245 10.374 14.051 −6.266 1.00 38.61 C ANISOU 67 CA PRO A 245 4913 4858 4895 −18 −64 −80 C ATOM 68 CB PRO A 245 9.108 13.286 −5.850 1.00 38.29 C ANISOU 68 CB PRO A 245 4913 4765 4870 −17 −31 −43 C ATOM 69 CG PRO A 245 7.963 13.883 −6.657 1.00 38.73 C ANISOU 69 CG PRO A 245 4943 4859 4914 −8 −25 −82 C ATOM 70 CD PRO A 245 8.576 14.533 −7.845 1.00 38.57 C ANISOU 70 CD PRO A 245 4919 4821 4912 41 −48 −19 C ATOM 71 C PRO A 245 11.490 13.073 −6.621 1.00 38.77 C ANISOU 71 C PRO A 245 4914 4890 4926 −38 −52 −39 C ATOM 72 O PRO A 245 11.863 12.917 −7.800 1.00 37.94 O ANISOU 72 O PRO A 245 4774 4779 4862 −114 −92 −2 O ATOM 73 N LYS A 246 12.028 12.430 −5.589 1.00 39.37 N ANISOU 73 N LYS A 246 4984 4983 4991 −16 −60 −30 N ATOM 74 CA LYS A 246 12.931 11.318 −5.796 1.00 39.82 C ANISOU 74 CA LYS A 246 5023 5058 5050 14 −26 −33 C ATOM 75 CB LYS A 246 13.669 10.947 −4.509 1.00 40.23 C ANISOU 75 CB LYS A 246 5091 5073 5120 18 −39 −40 C ATOM 76 CG LYS A 246 14.888 11.842 −4.290 1.00 41.46 C ANISOU 76 CG LYS A 246 5178 5330 5245 −27 39 8 C ATOM 77 CD LYS A 246 15.623 12.015 −5.627 1.00 45.52 C ANISOU 77 CD LYS A 246 5812 5935 5546 66 −16 −88 C ATOM 78 CE LYS A 246 16.445 13.285 −5.737 1.00 46.82 C ANISOU 78 CE LYS A 246 5686 5640 6463 −111 −80 −47 C ATOM 79 NZ LYS A 246 16.762 13.502 −7.165 1.00 46.41 N ANISOU 79 NZ LYS A 246 5883 6214 5535 18 173 183 N ATOM 80 C LYS A 246 12.159 10.148 −6.374 1.00 39.48 C ANISOU 80 C LYS A 246 4992 5034 4975 21 −27 −32 C ATOM 81 O LYS A 246 11.088 9.797 −5.861 1.00 39.69 O ANISOU 81 O LYS A 246 5028 5127 4923 65 −62 −106 O ATOM 82 N PRO A 247 12.697 9.540 −7.448 1.00 39.18 N ANISOU 82 N PRO A 247 4951 5000 4932 33 −13 −12 N ATOM 83 CA PRO A 247 12.000 8.478 −8.161 1.00 38.92 C ANISOU 83 CA PRO A 247 4924 4959 4905 51 −17 −16 C ATOM 84 CB PRO A 247 13.085 7.878 −9.054 1.00 38.79 C ANISOU 84 CB PRO A 247 4920 4940 4877 55 8 −7 C ATOM 85 CG PRO A 247 14.014 8.985 −9.285 1.00 38.59 C ANISOU 85 CG PRO A 247 4889 4914 4858 19 11 −18 C ATOM 86 CD PRO A 247 14.021 9.809 −8.040 1.00 39.18 C ANISOU 86 CD PRO A 247 4973 4976 4934 18 3 −6 C ATOM 87 C PRO A 247 11.450 7.425 −7.230 1.00 38.87 C ANISOU 87 C PRO A 247 4936 4933 4896 54 4 −38 C ATOM 88 O PRO A 247 10.303 7.039 −7.385 1.00 38.56 O ANISOU 88 O PRO A 247 4878 4910 4862 97 6 −34 O ATOM 89 N LYS A 248 12.246 6.970 −6.261 1.00 38.99 N ANISOU 89 N LYS A 248 4950 4955 4907 43 4 −29 N ATOM 90 CA LYS A 248 11.781 5.891 −5.389 1.00 38.78 C ANISOU 90 CA LYS A 248 4934 4936 4863 41 −23 −10 C ATOM 91 CB LYS A 248 12.937 5.021 −4.850 1.00 39.27 C ANISOU 91 CB LYS A 248 5026 4978 4917 7 −18 −25 C ATOM 92 CG LYS A 248 13.648 5.482 −3.580 1.00 36.38 C ANISOU 92 CG LYS A 248 4760 4251 4810 413 −171 220 C ATOM 93 CD LYS A 248 14.700 4.434 −3.086 1.00 40.67 C ANISOU 93 CD LYS A 248 5196 5302 4953 −77 72 −121 C ATOM 94 CE LYS A 248 14.061 3.085 −2.711 1.00 35.80 C ANISOU 94 CE LYS A 248 4447 4475 4680 291 −218 261 C ATOM 95 NZ LYS A 248 15.004 2.044 −2.179 1.00 40.36 N ANISOU 95 NZ LYS A 248 5159 5539 4635 −366 221 −488 N ATOM 96 C LYS A 248 10.799 6.352 −4.312 1.00 38.61 C ANISOU 96 C LYS A 248 4939 4906 4825 14 −19 −2 C ATOM 97 O LYS A 248 10.063 5.550 −3.775 1.00 38.84 O ANISOU 97 O LYS A 248 5003 4972 4783 16 −22 −36 O ATOM 98 N ASP A 249 10.750 7.655 −4.033 1.00 38.44 N ANISOU 98 N ASP A 249 4875 4955 4772 44 3 −16 N ATOM 99 CA ASP A 249 9.691 8.181 −3.171 1.00 37.92 C ANISOU 99 CA ASP A 249 4847 4859 4700 48 −19 22 C ATOM 100 CB ASP A 249 9.970 9.633 −2.774 1.00 38.25 C ANISOU 100 CB ASP A 249 4896 4868 4767 11 7 17 C ATOM 101 CG ASP A 249 10.882 9.741 −1.587 1.00 38.86 C ANISOU 101 CG ASP A 249 5031 4871 4860 18 23 22 C ATOM 102 OD1 ASP A 249 11.024 8.755 −0.860 1.00 41.27 O ANISOU 102 OD1 ASP A 249 5480 5121 5080 125 28 −54 O ATOM 103 OO2 ASP A 249 11.457 10.815 −1.352 1.00 41.70 O ANISOU 103 OD2 ASP A 249 5332 5260 5250 −57 123 4 O ATOM 104 C ASP A 249 8.325 8.043 −3.853 1.00 36.93 C ANISOU 104 C ASP A 249 4734 4748 4547 24 −9 14 C ATOM 105 O ASP A 249 7.300 7.842 −3.198 1.00 36.04 O ANISOU 105 O ASP A 249 4654 4626 4413 57 −57 34 O ATOM 106 N THR A 250 8.338 8.136 −5.182 1.00 36.24 N ANISOU 106 N THR A 250 4619 4672 4477 50 −15 16 N ATOM 107 CA THR A 250 7.131 7.982 −5.982 1.00 35.08 C ANISOU 107 CA THR A 250 4477 4569 4283 0 2 47 C ATOM 108 CB THR A 250 7.233 8.753 −7.345 1.00 35.01 C ANISOU 108 CB THR A 250 4440 4538 4324 26 1 31 C ATOM 109 OG1 THR A 250 7.969 7.992 −8.287 1.00 33.57 O ANISOU 109 OG1 THR A 250 4192 4584 3978 26 −86 100 O ATOM 110 CG2 THR A 250 7.901 10.112 −7.157 1.00 34.29 C ANISOU 110 CG2 THR A 250 4178 4535 4312 −61 −10 167 C ATOM 111 C THR A 250 6.674 6.521 −6.163 1.00 34.51 C ANISOU 111 C THR A 250 4501 4473 4136 12 33 −4 C ATOM 112 O THR A 250 5.499 6.269 −6.443 1.00 33.70 O ANISOU 112 O THR A 250 4487 4349 3968 −15 94 −34 O ATOM 113 N LEU A 251 7.570 5.569 −5.940 1.00 34.49 N ANISOU 113 N LEU A 251 4464 4483 4158 −14 57 −24 N ATOM 114 CA LEU A 251 7.288 4.145 −6.229 1.00 35.46 C ANISOU 114 CA LEU A 251 4591 4519 4360 −36 56 39 C ATOM 115 CB LEU A 251 8.416 3.552 −7.084 1.00 34.39 C ANISOU 115 CB LEU A 251 4522 4286 4258 −37 43 44 C ATOM 116 CG LEU A 251 8.640 4.234 −8.448 1.00 32.40 C ANISOU 116 CG LEU A 251 4144 4197 3968 −25 0 −41 C ATOM 117 CD1 LEU A 251 9.950 3.850 −9.025 1.00 31.29 C ANISOU 117 CD1 LEU A 251 4008 4153 3728 70 −120 25 C ATOM 118 CD2 LEU A 251 7.538 3.937 −9.435 1.00 30.02 C ANISOU 118 CD2 LEU A 251 3862 3832 3709 −19 204 164 C ATOM 119 C LEU A 251 6.967 3.247 −4.999 1.00 36.71 C ANISOU 119 C LEU A 251 4788 4688 4471 −34 12 51 C ATOM 120 O LEU A 251 6.530 2.097 −5.126 1.00 36.26 O ANISOU 120 O LEU A 251 4757 4604 4414 −55 47 142 O ATOM 121 N MET A 252 7.187 3.791 −3.816 1.00 38.60 N ANISOU 121 N MET A 252 5026 4943 4698 −27 20 29 N ATOM 122 CA MET A 252 6.895 3.102 −2.566 1.00 40.29 C ANISOU 122 CA MET A 252 5241 5198 4870 1 −7 44 C ATOM 123 CB MET A 252 8.114 3.092 −1.677 1.00 40.45 C ANISOU 123 CB MET A 252 5226 5262 4879 −22 −28 −14 C ATOM 124 CG MET A 252 9.210 2.179 −2.138 1.00 42.62 C ANISOU 124 CG MET A 252 5404 5525 5262 48 6 −36 C ATOM 125 SD MET A 252 10.657 2.547 −1.165 1.00 43.78 S ANISOU 125 SD MET A 252 5542 5849 5244 48 −85 63 S ATOM 126 CE MET A 252 10.140 1.837 0.396 1.00 45.33 C ANISOU 126 CE MET A 252 5743 5827 5653 −34 103 102 C ATOM 127 C MET A 252 5.829 3.879 −1.874 1.00 39.26 C ANISOU 127 C MET A 252 5153 5083 4681 −6 −19 52 C ATOM 128 O MET A 252 6.043 5.040 −1.508 1.00 39.40 O ANISOU 128 O MET A 252 5246 5129 4594 −46 −43 54 O ATOM 129 N ILE A 253 4.682 3.237 −1.700 1.00 39.08 N ANISOU 129 N ILE A 253 5163 5016 4667 12 −39 49 N ATOM 130 CA ILE A 253 3.486 3.888 −1.183 1.00 39.29 C ANISOU 130 CA ILE A 253 5103 5050 4775 29 12 19 C ATOM 131 CB ILE A 253 2.247 3.011 −1.395 1.00 38.95 C ANISOU 131 CB ILE A 253 5067 5028 4703 31 24 14 C ATOM 132 CG1 ILE A 253 0.953 3.823 −1.205 1.00 39.49 C ANISOU 132 CG1 ILE A 253 5092 5115 4795 9 −6 −38 C ATOM 133 CD1 ILE A 253 −0.330 3.042 −1.535 1.00 38.97 C ANISOU 133 CD1 ILE A 253 5004 5047 4756 10 47 50 C ATOM 134 CG2 ILE A 253 2.320 1.777 −0.515 1.00 39.92 C ANISOU 134 CG2 ILE A 253 5138 5135 4895 40 −36 1 C ATOM 135 C ILE A 253 3.622 4.327 0.280 1.00 39.88 C ANISOU 135 C ILE A 253 5183 5139 4829 56 53 44 C ATOM 136 O ILE A 253 2.795 5.089 0.776 1.00 40.73 O ANISOU 136 O ILE A 253 5278 5260 4936 23 76 42 O ATOM 137 N SER A 254 4.683 3.892 0.950 1.00 40.46 N ANISOU 137 N SER A 254 5255 5229 4888 63 59 23 N ATOM 138 CA SER A 254 4.863 4.181 2.364 1.00 40.63 C ANISOU 138 CA SER A 254 5276 5248 4914 9 50 −9 C ATOM 139 CB SER A 254 5.567 3.005 3.055 1.00 40.37 C ANISOU 139 CB SER A 254 5254 5212 4870 59 53 −23 C ATOM 140 OG SER A 254 6.984 3.119 2.972 1.00 40.90 O ANISOU 140 OG SER A 254 5421 5224 4893 −34 108 −21 O ATOM 141 C SER A 254 5.628 5.488 2.550 1.00 40.97 C ANISOU 141 C SER A 254 5312 5271 4982 −19 76 25 C ATOM 142 O SER A 254 5.603 6.114 3.632 1.00 41.28 O ANISOU 142 O SER A 254 5346 5328 5007 −41 116 79 O ATOM 143 N ARG A 255 6.307 5.897 1.484 1.00 41.20 N ANISOU 143 N ARG A 255 5344 5362 4946 −17 73 −22 N ATOM 144 CA ARG A 255 7.116 7.128 1.464 1.00 40.77 C ANISOU 144 CA ARG A 255 5217 5295 4977 −30 15 −71 C ATOM 145 CB ARG A 255 8.312 6.960 0.520 1.00 41.05 C ANISOU 145 CB ARG A 255 5254 5318 5025 −26 19 −59 C ATOM 146 CG ARG A 255 9.203 5.754 0.873 1.00 41.23 C ANISOU 146 CG ARG A 255 5241 5314 5109 −16 −53 −75 C ATOM 147 CD ARG A 255 10.479 5.781 0.055 1.00 42.71 C ANISOU 147 CD ARG A 255 5282 5481 5463 −20 −70 −52 C ATOM 148 NE ARG A 255 11.486 4.874 0.595 1.00 44.95 N ANISOU 148 NE ARG A 255 5686 5632 5761 −23 −8 6 N ATOM 149 CZ ARG A 255 12.800 5.096 0.570 1.00 44.62 C ANISOU 149 CZ ARG A 255 5581 5638 5732 −42 −75 37 C ATOM 150 NH1 ARG A 255 13.283 6.213 0.045 1.00 44.95 N ANISOU 150 NH1 ARG A 255 5600 5866 5612 −153 −140 84 N ATOM 151 NH2 ARG A 255 13.632 4.205 1.093 1.00 45.14 N ANISOU 151 NH2 ARG A 255 5708 5780 5660 6 −107 −75 N ATOM 152 C ARG A 255 6.300 8.371 1.111 1.00 40.62 C ANISOU 152 C ARG A 255 5207 5295 4932 −38 26 −78 C ATOM 153 O ARG A 255 5.092 8.307 0.939 1.00 41.23 O ANISOU 153 O ARG A 255 5213 5455 4994 0 −1 −130 O ATOM 154 N THR A 256 6.959 9.512 1.013 1.00 40.48 N ANISOU 154 N THR A 256 5221 5247 4910 −13 43 −73 N ATOM 155 CA THR A 256 6.253 10.779 0.949 1.00 40.65 C ANISOU 155 CA THR A 256 5233 5241 4969 29 14 6 C ATOM 156 CB THR A 256 6.265 11.522 2.350 1.00 40.92 C ANISOU 156 CB THR A 256 5291 5223 5035 40 47 −14 C ATOM 157 OG1 THR A 256 6.218 10.571 3.438 1.00 42.34 O ANISOU 157 OG1 THR A 256 5543 5436 5106 142 62 −151 O ATOM 158 CG2 THR A 256 5.082 12.466 2.479 1.00 41.51 C ANISOU 158 CG2 THR A 256 5386 5196 5190 75 11 15 C ATOM 159 C THR A 256 6.931 11.637 −0.116 1.00 40.11 C ANISOU 159 C THR A 256 5160 5179 4899 0 8 30 C ATOM 160 O THR A 256 7.896 12.347 0.183 1.00 40.28 O ANISOU 160 O THR A 256 5245 5223 4835 −24 20 133 O ATOM 161 N PRO A 257 6.448 11.568 −1.366 1.00 39.60 N ANISOU 161 N PRO A 257 5072 5102 4872 −11 3 −7 N ATOM 162 CA PRO A 257 7.093 12.373 −2.402 1.00 39.43 C ANISOU 162 CA PRO A 257 5067 5077 4836 1 −29 −23 C ATOM 163 CB PRO A 257 6.587 11.750 −3.707 1.00 39.18 C ANISOU 163 CB PRO A 257 5053 5055 4778 7 −3 −63 C ATOM 164 CG PRO A 257 5.294 11.091 −3.363 1.00 39.06 C ANISOU 164 CG PRO A 257 5063 5013 4764 −13 −1 −34 C ATOM 165 CD PRO A 257 5.315 10.781 −1.890 1.00 40.06 C ANISOU 165 CD PRO A 257 5172 5085 4963 −7 17 5 C ATOM 166 C PRO A 257 6.744 13.855 −2.321 1.00 39.42 C ANISOU 166 C PRO A 257 5113 5073 4792 −38 −37 −56 C ATOM 167 O PRO A 257 5.645 14.229 −1.926 1.00 38.86 O ANISOU 167 O PRO A 257 5152 4918 4693 −21 −22 −87 O ATOM 168 N GLU A 258 7.692 14.683 −2.730 1.00 40.26 N ANISOU 168 N GLU A 258 5231 5155 4908 −57 −80 −131 N ATOM 169 CA GLU A 258 7.562 16.117 −2.647 1.00 40.63 C ANISOU 169 CA GLU A 258 5252 5182 5001 −31 −77 −91 C ATOM 170 CB GLU A 258 8.386 16.633 −1.456 1.00 41.53 C ANISOU 170 CB GLU A 258 5416 5269 5091 −15 −86 −96 C ATOM 171 CG GLU A 258 7.818 16.238 −0.078 1.00 44.60 C ANISOU 171 CG GLU A 258 5886 5562 5495 −57 188 −51 C ATOM 172 CD GLU A 258 8.897 16.116 1.007 1.00 40.88 C ANISOU 172 CD GLU A 258 4965 5716 4850 −392 −49 386 C ATOM 173 OE1 GLU A 258 8.594 15.520 2.079 1.00 48.80 O ANISOU 173 OE1 GLU A 258 6168 6166 6208 50 −119 −284 O ATOM 174 OE2 GLU A 258 10.041 16.600 0.785 1.00 46.91 O ANISOU 174 OE2 GLU A 258 6298 5858 5666 75 −119 −191 O ATOM 175 C GLU A 258 8.092 16.753 −3.903 1.00 40.29 C ANISOU 175 C GLU A 258 5202 5119 4985 −31 −97 −129 C ATOM 176 O GLU A 258 9.072 16.292 −4.477 1.00 40.44 O ANISOU 176 O GLU A 258 5293 5116 4956 −54 −98 −185 O ATOM 177 N VAL A 259 7.459 17.840 −4.303 1.00 40.01 N ANISOU 177 N VAL A 259 5143 5064 4994 −48 −92 −112 N ATOM 178 CA VAL A 259 7.948 18.670 −5.392 1.00 40.26 C ANISOU 178 CA VAL A 259 5155 5061 5081 18 −77 −86 C ATOM 179 CB VAL A 259 6.797 18.967 −6.381 1.00 40.32 C ANISOU 179 CB VAL A 259 5194 5086 5040 3 −70 −79 C ATOM 180 CG1 VAL A 259 7.169 20.031 −7.349 1.00 39.85 C ANISOU 180 CG1 VAL A 259 5102 5046 4994 −46 −49 −51 C ATOM 181 CG2 VAL A 259 6.390 17.682 −7.103 1.00 40.01 C ANISOU 181 CG2 VAL A 259 5111 5024 5065 55 −92 −113 C ATOM 182 C VAL A 259 8.529 19.942 −4.761 1.00 40.31 C ANISOU 182 C VAL A 259 5146 5030 5137 22 −59 −6 C ATOM 183 O VAL A 259 7.939 20.517 −3.840 1.00 39.93 O ANISOU 183 O VAL A 259 5181 4910 5080 30 −43 −21 O ATOM 184 N THR A 260 9.704 20.355 −5.211 1.00 40.59 N ANISOU 184 N THR A 260 5198 5044 5180 37 −40 1 N ATOM 185 CA THR A 260 10.377 21.475 −4.556 1.00 40.90 C ANISOU 185 CA THR A 260 5225 5103 5210 17 −22 −52 C ATOM 186 CB THR A 260 11.722 21.052 −3.918 1.00 40.60 C ANISOU 186 CB THR A 260 5177 5055 5191 5 7 −62 C ATOM 187 OG1 THR A 260 11.488 19.986 −2.986 1.00 40.40 O ANISOU 187 OG1 THR A 260 5213 5077 5059 47 26 −208 O ATOM 188 CG2 THR A 260 12.342 22.196 −3.157 1.00 40.61 C ANISOU 188 CG2 THR A 260 5192 5102 5132 −11 38 10 C ATOM 189 C THR A 260 10.510 22.656 −5.512 1.00 41.22 C ANISOU 189 C THR A 260 5246 5148 5267 −11 −3 −54 C ATOM 190 O THR A 260 11.068 22.527 −6.598 1.00 41.63 O ANISOU 190 O THR A 260 5257 5264 5294 15 23 −122 O ATOM 191 N CYS A 261 9.943 23.795 −5.109 1.00 41.33 N ANISOU 191 N CYS A 261 5272 5136 5296 −17 −27 −88 N ATOM 192 CA CYS A 261 10.029 25.032 −5.889 1.00 41.26 C ANISOU 192 CA CYS A 261 5292 5146 5237 −26 −50 −63 C ATOM 193 CB CYS A 261 8.691 25.769 −5.894 1.00 40.72 C ANISOU 193 CB CYS A 261 5249 5103 5116 −4 −33 −99 C ATOM 194 SG CYS A 261 8.495 27.040 −7.213 1.00 41.29 S ANISOU 194 SG CYS A 261 5307 4989 5391 −25 −29 −151 S ATOM 195 C CYS A 261 11.104 25.928 −5.292 1.00 41.60 C ANISOU 195 C CYS A 261 5340 5207 5259 −19 −18 −67 C ATOM 196 O CYS A 261 11.014 26.324 −4.133 1.00 42.19 O ANISOU 196 O CYS A 261 5452 5297 5280 −24 −69 −78 O ATOM 197 N VAL A 262 12.121 26.234 −6.084 1.00 41.65 N ANISOU 197 N VAL A 262 5287 5236 5301 −27 5 −70 N ATOM 198 CA VAL A 262 13.192 27.102 −5.645 1.00 41.52 C ANISOU 198 CA VAL A 262 5296 5172 5305 −10 −8 −79 C ATOM 199 CB VAL A 262 14.566 26.403 −5.736 1.00 41.57 C ANISOU 199 CB VAL A 262 5266 5213 5312 −4 −11 −73 C ATOM 200 CG1 VAL A 262 15.703 27.418 −5.531 1.00 41.51 C ANISOU 200 CG1 VAL A 262 5318 5058 5393 16 35 −129 C ATOM 201 CG2 VAL A 262 14.667 25.238 −4.732 1.00 40.60 C ANISOU 201 CG2 VAL A 262 5239 5053 5131 −58 −15 −107 C ATOM 202 C VAL A 262 13.230 28.375 −6.493 1.00 41.97 C ANISOU 202 C VAL A 262 5353 5249 5344 −6 1 −62 C ATOM 203 O VAL A 262 13.277 28.308 −7.744 1.00 41.34 O ANISOU 203 O VAL A 262 5297 5185 5225 3 27 −162 O ATOM 204 N VAL A 263 13.200 29.523 −5.795 1.00 41.79 N ANISOU 204 N VAL A 263 5323 5198 5357 −8 26 −81 N ATOM 205 CA VAL A 263 13.450 30.829 −6.399 1.00 41.87 C ANISOU 205 CA VAL A 263 5331 5210 5365 13 −1 −48 C ATOM 206 CB VAL A 263 12.381 31.882 −6.011 1.00 42.15 C ANISOU 206 CB VAL A 263 5365 5244 5407 −13 −13 −29 C ATOM 207 CG1 VAL A 263 12.197 32.897 −7.151 1.00 41.64 C ANISOU 207 CG1 VAL A 263 5414 5099 5308 −3 −61 −36 C ATOM 208 CG2 VAL A 263 11.070 31.217 −5.688 1.00 41.69 C ANISOU 208 CG2 VAL A 263 5219 5254 5366 28 11 9 C ATOM 209 C VAL A 263 14.825 31.331 −5.954 1.00 42.07 C ANISOU 209 C VAL A 263 5367 5246 5371 −13 24 −47 C ATOM 210 O VAL A 263 15.194 31.243 −4.773 1.00 42.24 O ANISOU 210 O VAL A 263 5419 5263 5365 −9 12 −77 O ATOM 211 N VAL A 264 15.594 31.819 −6.915 1.00 42.34 N ANISOU 211 N VAL A 264 5407 5261 5417 −40 15 −20 N ATOM 212 CA VAL A 264 16.912 32.371 −6.640 1.00 42.34 C ANISOU 212 CA VAL A 264 5355 5321 5408 −44 −15 0 C ATOM 213 CB VAL A 264 18.086 31.458 −7.123 1.00 42.32 C ANISOU 213 CB VAL A 264 5369 5281 5427 −74 −22 5 C ATOM 214 CG1 VAL A 264 18.307 30.333 −6.151 1.00 42.89 C ANISOU 214 CG1 VAL A 264 5398 5442 5455 11 −96 65 C ATOM 215 CG2 VAL A 264 17.862 30.926 −8.536 1.00 42.03 C ANISOU 215 CG2 VAL A 264 5346 5351 5270 −55 −7 25 C ATOM 216 C VAL A 264 17.003 33.756 −7.271 1.00 42.94 C ANISOU 216 C VAL A 264 5419 5400 5493 −38 −13 22 C ATOM 217 O VAL A 264 16.131 34.135 −8.077 1.00 43.29 O ANISOU 217 O VAL A 264 5396 5527 5525 −14 −27 41 O ATOM 218 N ASP A 265 18.057 34.500 −6.918 1.00 42.97 N ANISOU 218 N ASP A 265 5466 5374 5487 −58 −15 −6 N ATOM 219 CA ASP A 265 18.204 35.890 −7.353 1.00 43.20 C ANISOU 219 CA ASP A 265 5502 5401 5511 −43 11 9 C ATOM 220 CB ASP A 265 18.142 36.018 −8.889 1.00 43.15 C ANISOU 220 CB ASP A 265 5545 5394 5456 −50 −12 −19 C ATOM 221 CG ASP A 265 19.371 35.453 −9.579 1.00 44.92 C ANISOU 221 CG ASP A 265 5679 5711 5678 −48 12 −4 C ATOM 222 OD1 ASP A 265 19.303 35.191 −10.803 1.00 46.63 O ANISOU 222 OD1 ASP A 265 5857 6021 5836 −140 −10 −71 O ATOM 223 OD2 ASP A 265 20.411 35.263 −8.906 1.00 47.21 O ANISOU 223 OD2 ASP A 265 5935 5986 6016 −141 −96 −22 O ATOM 224 C ASP A 265 17.117 36.728 −6.695 1.00 43.08 C ANISOU 224 C ASP A 265 5496 5395 5475 −37 49 −4 C ATOM 225 O ASP A 265 16.547 37.636 −7.313 1.00 43.22 O ANISOU 225 O ASP A 265 5505 5422 5495 −37 102 40 O ATOM 226 N VAL A 266 16.787 36.397 −5.449 1.00 43.36 N ANISOU 226 N VAL A 266 5544 5410 5520 −42 9 −52 N ATOM 227 CA VAL A 266 15.823 37.227 −4.722 1.00 43.59 C ANISOU 227 CA VAL A 266 5576 5445 5541 −38 −7 −62 C ATOM 228 CB VAL A 266 15.117 36.476 −3.564 1.00 43.62 C ANISOU 228 CB VAL A 266 5567 5440 5564 −36 −10 −65 C ATOM 229 CG1 VAL A 266 14.260 37.426 −2.730 1.00 43.27 C ANISOU 229 CG1 VAL A 266 5518 5469 5453 −20 −46 −125 C ATOM 230 CG2 VAL A 266 14.253 35.309 −4.109 1.00 43.36 C ANISOU 230 CG2 VAL A 266 5463 5462 5547 25 −14 −83 C ATOM 231 C VAL A 266 16.653 38.421 −4.250 1.00 43.81 C ANISOU 231 C VAL A 266 5612 5461 5572 −40 −47 −75 C ATOM 232 O VAL A 266 17.678 38.243 −3.606 1.00 44.05 O ANISOU 232 O VAL A 266 5645 5485 5605 −62 −80 −95 O ATOM 233 N SER A 267 16.252 39.625 −4.629 1.00 44.16 N ANISOU 233 N SER A 267 5674 5467 5637 −42 −51 −55 N ATOM 234 CA SER A 267 17.044 40.809 −4.291 1.00 44.92 C ANISOU 234 CA SER A 267 5742 5605 5720 −26 −25 −27 C ATOM 235 CB SER A 267 16.574 42.015 −5.098 1.00 44.80 C ANISOU 235 CB SER A 267 5731 5551 5737 −24 −13 −6 C ATOM 236 OG SER A 267 15.463 42.605 −4.458 1.00 44.95 O ANISOU 236 OG SER A 267 5676 5584 5816 9 4 −13 O ATOM 237 C SER A 267 16.978 41.119 −2.789 1.00 45.15 C ANISOU 237 C SER A 267 5807 5630 5718 −32 −6 −55 C ATOM 238 O SER A 267 16.586 40.278 −1.978 1.00 45.49 O ANISOU 238 O SER A 267 5828 5712 5744 −51 7 −60 O ATOM 239 N HIS A 268 17.366 42.333 −2.428 1.00 45.94 N ANISOU 239 N HIS A 268 5897 5755 5801 −37 −7 −42 N ATOM 240 CA HIS A 268 17.252 42.793 −1.044 1.00 46.15 C ANISOU 240 CA HIS A 268 5932 5783 5817 −26 −11 −31 C ATOM 241 CB HIS A 268 18.614 43.221 −0.546 1.00 46.45 C ANISOU 241 CB HIS A 268 5950 5843 5855 −51 −45 −21 C ATOM 242 CG HIS A 268 19.575 42.093 −0.372 1.00 47.75 C ANISOU 242 CG HIS A 268 6131 5941 6069 30 −23 −45 C ATOM 243 ND1 HIS A 268 19.941 41.617 0.869 1.00 49.83 N ANISOU 243 ND1 HIS A 268 6445 6227 6259 116 7 −9 N ATOM 244 CE1 HIS A 268 20.804 40.628 0.722 1.00 49.57 C ANISOU 244 CE1 HIS A 268 6470 6345 6018 34 60 −31 C ATOM 245 NE2 HIS A 268 21.011 40.445 −0.570 1.00 49.91 N ANISOU 245 NE2 HIS A 268 6326 6330 6306 20 −64 2 N ATOM 246 CD2 HIS A 268 20.257 41.354 −1.276 1.00 48.94 C ANISOU 246 CD2 HIS A 268 6300 6208 6085 12 8 −40 C ATOM 247 C HIS A 268 16.270 43.953 −0.932 1.00 46.31 C ANISOU 247 C HIS A 268 5952 5787 5855 −19 8 −26 C ATOM 248 O HIS A 268 15.635 44.151 0.120 1.00 46.57 O ANISOU 248 O HIS A 268 6023 5813 5856 −91 5 −43 O ATOM 249 N GLU A 269 16.140 44.701 −2.032 1.00 46.57 N ANISOU 249 N GLU A 269 5987 5794 5912 0 24 16 N ATOM 250 CA GLU A 269 15.251 45.875 −2.109 1.00 46.95 C ANISOU 250 CA GLU A 269 5979 5849 6010 4 29 −27 C ATOM 251 CB GLU A 269 15.603 46.754 −3.323 1.00 46.62 C ANISOU 251 CB GLU A 269 5977 5776 5960 −3 18 −3 C ATOM 252 CG GLU A 269 17.070 46.679 −3.764 1.00 47.69 C ANISOU 252 CG GLU A 269 5996 5921 6202 −64 33 27 C ATOM 253 CD GLU A 269 17.958 47.749 −3.127 1.00 50.37 C ANISOU 253 CD GLU A 269 6386 6291 6460 −14 −39 −64 C ATOM 254 OE1 GLU A 269 17.419 48.766 −2.634 1.00 49.63 O ANISOU 254 OE1 GLU A 269 6277 6189 6390 40 −9 −219 O ATOM 255 OE2 GLU A 269 19.208 47.585 −3.161 1.00 51.70 O ANISOU 255 OE2 GLU A 269 6319 6463 6858 16 −192 20 O ATOM 256 C GLU A 269 13.797 45.428 −2.211 1.00 47.24 C ANISOU 256 C GLU A 269 6001 5892 6054 2 38 −55 C ATOM 257 O GLU A 269 12.901 46.013 −1.583 1.00 48.33 O ANISOU 257 O GLU A 269 6161 6020 6182 27 66 −84 O ATOM 258 N ASP A 270 13.572 44.392 −3.019 1.00 47.26 N ANISOU 258 N ASP A 270 6029 5892 6034 10 36 −52 N ATOM 259 CA ASP A 270 12.251 43.806 −3.226 1.00 46.94 C ANISOU 259 CA ASP A 270 5979 5862 5992 11 11 −19 C ATOM 260 CB ASP A 270 11.817 44.010 −4.678 1.00 47.64 C ANISOU 260 CB ASP A 270 6125 5924 6052 −10 2 −55 C ATOM 261 CG ASP A 270 12.011 45.446 −5.149 1.00 49.53 C ANISOU 261 CG ASP A 270 6428 6106 6283 5 83 46 C ATOM 262 OD1 ASP A 270 11.562 46.383 −4.426 1.00 50.58 O ANISOU 262 OD1 ASP A 270 6655 6105 6455 75 138 −47 O ATOM 263 OD2 ASP A 270 12.620 45.632 −6.232 1.00 50.17 O ANISOU 263 OD2 ASP A 270 6461 6333 6269 47 198 −153 O ATOM 264 C ASP A 270 12.379 42.331 −2.925 1.00 46.46 C ANISOU 264 C ASP A 270 5904 5821 5928 4 5 −41 C ATOM 265 O ASP A 270 12.490 41.528 −3.852 1.00 46.73 O ANISOU 265 O ASP A 270 5861 5902 5992 73 80 −3 O ATOM 266 N PRO A 271 12.383 41.970 −1.628 1.00 46.30 N ANISOU 266 N PRO A 271 5872 5815 5902 19 9 −39 N ATOM 267 CA PRO A 271 12.735 40.625 −1.182 1.00 46.38 C ANISOU 267 CA PRO A 271 5878 5802 5940 8 8 −53 C ATOM 268 CB PRO A 271 13.321 40.873 0.207 1.00 46.74 C ANISOU 268 CB PRO A 271 5927 5842 5990 0 20 −18 C ATOM 269 CG PRO A 271 12.532 42.082 0.732 1.00 46.35 C ANISOU 269 CG PRO A 271 5868 5817 5922 37 −13 −51 C ATOM 270 CD PRO A 271 12.074 42.863 −0.487 1.00 46.34 C ANISOU 270 CD PRO A 271 5887 5812 5907 47 16 −33 C ATOM 271 C PRO A 271 11.567 39.661 −1.064 1.00 46.91 C ANISOU 271 C PRO A 271 5915 5843 6064 16 18 −55 C ATOM 272 O PRO A 271 11.751 38.464 −1.297 1.00 47.80 O ANISOU 272 O PRO A 271 5999 5932 6231 72 10 −41 O ATOM 273 N GLU A 272 10.385 40.161 −0.703 1.00 47.03 N ANISOU 273 N GLU A 272 5924 5834 6111 10 9 −81 N ATOM 274 CA GLU A 272 9.255 39.289 −0.375 1.00 46.90 C ANISOU 274 CA GLU A 272 5899 5861 6056 −31 2 −69 C ATOM 275 CB GLU A 272 8.199 40.002 0.480 1.00 47.05 C ANISOU 275 CB GLU A 272 5905 5909 6061 −29 −1 −57 C ATOM 276 CG GLU A 272 8.736 40.972 1.528 1.00 48.65 C ANISOU 276 CG GLU A 272 6104 6206 6174 −35 −24 −64 C ATOM 277 CD GLU A 272 9.674 40.329 2.566 1.00 50.71 C ANISOU 277 CD GLU A 272 6405 6393 6466 10 −66 39 C ATOM 278 OE1 GLU A 272 9.577 39.104 2.812 1.00 51.33 O ANISOU 278 OE1 GLU A 272 6528 6257 6717 −21 33 −12 O ATOM 279 OE2 GLU A 272 10.509 41.074 3.139 1.00 50.98 O ANISOU 279 OE2 GLU A 272 6448 6560 6361 −33 −177 −55 O ATOM 280 C GLU A 272 8.631 38.686 −1.633 1.00 46.54 C ANISOU 280 C GLU A 272 5881 5811 5989 −31 −30 −51 C ATOM 281 O GLU A 272 8.366 39.380 −2.630 1.00 47.00 O ANISOU 281 O GLU A 272 5929 5827 6100 −69 −26 −34 O ATOM 282 N VAL A 273 8.434 37.372 −1.573 1.00 45.97 N ANISOU 282 N VAL A 273 5822 5768 5875 −28 −35 −72 N ATOM 283 CA VAL A 273 7.935 36.586 −2.697 1.00 45.26 C ANISOU 283 CA VAL A 273 5738 5675 5781 −15 −4 −87 C ATOM 284 CB VAL A 273 9.042 35.675 −3.296 1.00 45.49 C ANISOU 284 CB VAL A 273 5723 5777 5781 −37 9 −110 C ATOM 285 CG1 VAL A 273 8.629 35.110 −4.669 1.00 45.42 C ANISOU 285 CG1 VAL A 273 5681 5791 5783 −59 0 −59 C ATOM 286 CG2 VAL A 273 10.363 36.439 −3.429 1.00 45.73 C ANISOU 286 CG2 VAL A 273 5844 5761 5769 −107 2 −86 C ATOM 287 C VAL A 273 6.749 35.756 −2.202 1.00 44.66 C ANISOU 287 C VAL A 273 5681 5592 5694 0 3 −104 C ATOM 288 O VAL A 273 6.687 35.353 −1.029 1.00 44.72 O ANISOU 288 O VAL A 273 5778 5509 5704 26 36 −129 O ATOM 289 N LYS A 274 5.811 35.522 −3.108 1.00 43.97 N ANISOU 289 N LYS A 274 5556 5534 5616 10 13 −109 N ATOM 290 CA LYS A 274 4.568 34.833 −2.807 1.00 43.21 C ANISOU 290 CA LYS A 274 5485 5435 5495 −5 21 −75 C ATOM 291 CB LYS A 274 3.407 35.759 −3.179 1.00 42.77 C ANISOU 291 CB LYS A 274 5436 5394 5419 0 −19 −102 C ATOM 292 CG LYS A 274 2.050 35.407 −2.597 1.00 41.82 C ANISOU 292 CG LYS A 274 5335 5189 5365 −22 −50 −111 C ATOM 293 CD LYS A 274 0.997 36.393 −3.092 1.00 42.08 C ANISOU 293 CD LYS A 274 5342 5275 5370 −13 −1 −45 C ATOM 294 CE LYS A 274 0.899 36.385 −4.614 1.00 44.59 C ANISOU 294 CE LYS A 274 5681 5789 5470 −483 152 −326 C ATOM 295 NZ LYS A 274 −0.045 37.398 −5.185 1.00 40.06 N ANISOU 295 NZ LYS A 274 5019 4610 5590 369 −248 81 N ATOM 296 C LYS A 274 4.498 33.570 −3.651 1.00 43.36 C ANISOU 296 C LYS A 274 5514 5454 5504 −3 30 −52 C ATOM 297 O LYS A 274 4.678 33.627 −4.873 1.00 43.92 O ANISOU 297 O LYS A 274 5587 5567 5534 35 17 −66 O ATOM 298 N PHE A 275 4.229 32.431 −3.024 1.00 43.43 N ANISOU 298 N PHE A 275 5510 5465 5523 17 5 −41 N ATOM 299 CA PHE A 275 4.007 31.206 −3.803 1.00 43.22 C ANISOU 299 CA PHE A 275 5474 5466 5480 9 9 −42 C ATOM 300 CB PHE A 275 4.673 30.013 −3.158 1.00 43.00 C ANISOU 300 CB PHE A 275 5486 5398 5452 2 15 −33 C ATOM 301 CG PHE A 275 6.166 30.040 −3.246 1.00 43.42 C ANISOU 301 CG PHE A 275 5474 5474 5547 30 −1 −25 C ATOM 302 CD1 PHE A 275 6.929 30.554 −2.193 1.00 42.75 C ANISOU 302 CD1 PHE A 275 5315 5360 5568 9 −25 −65 C ATOM 303 CE1 PHE A 275 8.322 30.570 −2.263 1.00 42.60 C ANISOU 303 CE1 PHE A 275 5337 5322 5527 −92 −56 14 C ATOM 304 CZ PHE A 275 8.962 30.062 −3.392 1.00 42.99 C ANISOU 304 CZ PHE A 275 5390 5492 5450 −41 −24 −57 C ATOM 305 CE2 PHE A 275 8.208 29.543 −4.459 1.00 43.86 C ANISOU 305 CE2 PHE A 275 5445 5657 5560 −71 26 −48 C ATOM 306 CD2 PHE A 275 6.818 29.538 −4.379 1.00 43.43 C ANISOU 306 CD2 PHE A 275 5481 5591 5428 9 −44 3 C ATOM 307 C PHE A 275 2.544 30.902 −4.001 1.00 43.40 C ANISOU 307 C PHE A 275 5526 5484 5479 −33 19 −47 C ATOM 308 O PHE A 275 1.750 30.906 −3.046 1.00 44.16 O ANISOU 308 O PHE A 275 5666 5605 5505 −26 30 −41 O ATOM 309 N ASN A 276 2.177 30.660 −5.252 1.00 43.24 N ANISOU 309 N ASN A 276 5500 5462 5466 −18 42 −22 N ATOM 310 CA ASN A 276 0.890 30.052 −5.539 1.00 43.32 C ANISOU 310 CA ASN A 276 5497 5427 5532 −7 31 −42 C ATOM 311 CB ASN A 276 0.089 30.864 −6.573 1.00 42.83 C ANISOU 311 CB ASN A 276 5423 5396 5453 32 70 −1 C ATOM 312 CG ASN A 276 −0.109 32.337 −6.171 1.00 43.44 C ANISOU 312 CG ASN A 276 5519 5499 5487 9 9 −16 C ATOM 313 OD1 ASN A 276 −0.722 33.112 −6.918 1.00 44.20 O ANISOU 313 OD1 ASN A 276 5682 5657 5455 −11 91 56 O ATOM 314 ND2 ASN A 276 0.392 32.724 −4.997 1.00 44.64 N ANISOU 314 ND2 ASN A 276 5694 5621 5644 84 −43 23 N ATOM 315 C ASN A 276 1.193 28.629 −6.021 1.00 43.23 C ANISOU 315 C ASN A 276 5471 5387 5567 12 67 −36 C ATOM 316 O ASN A 276 2.271 28.381 −6.566 1.00 43.45 O ANISOU 316 O ASN A 276 5468 5431 5610 15 74 −38 O ATOM 317 N TRP A 277 0.261 27.703 −5.780 1.00 43.29 N ANISOU 317 N TRP A 277 5473 5373 5600 17 43 −43 N ATOM 318 CA TRP A 277 0.403 26.290 −6.173 1.00 42.95 C ANISOU 318 CA TRP A 277 5415 5377 5523 −6 0 −103 C ATOM 319 CB TRP A 277 0.758 25.397 −4.985 1.00 42.16 C ANISOU 319 CB TRP A 277 5263 5304 5451 28 −4 −65 C ATOM 320 CG TRP A 277 2.183 25.417 −4.469 1.00 42.99 C ANISOU 320 CG TRP A 277 5406 5401 5527 5 23 −87 C ATOM 321 CD1 TRP A 277 2.657 26.129 −3.398 1.00 42.90 C ANISOU 321 CD1 TRP A 277 5338 5415 5547 10 −15 −143 C ATOM 322 NE1 TRP A 277 3.990 25.862 −3.194 1.00 43.36 N ANISOU 322 NE1 TRP A 277 5319 5500 5653 −54 −62 −48 N ATOM 323 CE2 TRP A 277 4.405 24.949 −4.125 1.00 42.25 C ANISOU 323 CE2 TRP A 277 5236 5343 5471 68 39 −134 C ATOM 324 CD2 TRP A 277 3.289 24.634 −4.943 1.00 42.84 C ANISOU 324 CD2 TRP A 277 5364 5403 5509 30 27 −96 C ATOM 325 CE3 TRP A 277 3.455 23.715 −5.991 1.00 43.02 C ANISOU 325 CE3 TRP A 277 5359 5403 5582 49 −27 −100 C ATOM 326 CZ3 TRP A 277 4.717 23.141 −6.186 1.00 42.65 C ANISOU 326 CZ3 TRP A 277 5369 5373 5462 −37 −12 −164 C ATOM 327 CH2 TRP A 277 5.801 23.471 −5.354 1.00 42.95 C ANISOU 327 CH2 TRP A 277 5324 5462 5533 8 −25 −87 C ATOM 328 CZ2 TRP A 277 5.668 24.376 −4.322 1.00 43.40 C ANISOU 328 CZ2 TRP A 277 5423 5476 5590 −13 −3 −46 C ATOM 329 C TRP A 277 −0.922 25.793 −6.720 1.00 42.96 C ANISOU 329 C TRP A 277 5381 5417 5524 9 0 −104 C ATOM 330 O TRP A 277 −1.978 26.129 −6.192 1.00 43.23 O ANISOU 330 O TRP A 277 5408 5439 5576 −15 −28 −119 O ATOM 331 N TYR A 278 −0.842 24.965 −7.754 1.00 42.84 N ANISOU 331 N TYR A 278 5366 5431 5478 38 −13 −101 N ATOM 332 CA TYR A 278 −1.988 24.432 −8.471 1.00 42.52 C ANISOU 332 CA TYR A 278 5338 5365 5451 19 −12 −52 C ATOM 333 CB TYR A 278 −2.247 25.265 −9.746 1.00 43.43 C ANISOU 333 CB TYR A 278 5419 5517 5565 0 −11 −30 C ATOM 334 CG TYR A 278 −2.307 26.769 −9.500 1.00 44.08 C ANISOU 334 CG TYR A 278 5578 5473 5695 8 54 0 C ATOM 335 CD1 TYR A 278 −1.140 27.535 −9.452 1.00 43.87 C ANISOU 335 CD1 TYR A 278 5454 5517 5694 19 −5 −65 C ATOM 336 CE1 TYR A 278 −1.177 28.900 −9.228 1.00 44.77 C ANISOU 336 CE1 TYR A 278 5712 5565 5732 0 −41 −53 C ATOM 337 CZ TYR A 278 −2.395 29.528 −9.019 1.00 44.46 C ANISOU 337 CZ TYR A 278 5561 5435 5893 3 −8 −83 C ATOM 338 OH TYR A 278 −2.429 30.889 −8.788 1.00 45.34 O ANISOU 338 OH TYR A 278 5756 5539 5929 47 81 −4 O ATOM 339 CE2 TYR A 278 −3.575 28.797 −9.047 1.00 46.00 C ANISOU 339 CE2 TYR A 278 5736 5868 5872 2 3 28 C ATOM 340 CD2 TYR A 278 −3.525 27.416 −9.291 1.00 44.42 C ANISOU 340 CD2 TYR A 278 5603 5446 5827 91 −7 −3 C ATOM 341 C TYR A 278 −1.705 22.978 −8.845 1.00 42.48 C ANISOU 341 C TYR A 278 5350 5371 5416 −2 −44 −14 C ATOM 342 O TYR A 278 −0.649 22.659 −9.386 1.00 42.02 O ANISOU 342 O TYR A 278 5346 5257 5361 43 −47 −56 O ATOM 343 N VAL A 279 −2.658 22.100 −8.551 1.00 42.92 N ANISOU 343 N VAL A 279 5472 5419 5416 −5 −44 20 N ATOM 344 CA VAL A 279 −2.610 20.701 −8.983 1.00 42.90 C ANISOU 344 CA VAL A 279 5447 5421 5431 20 −27 −3 C ATOM 345 CB VAL A 279 −2.902 19.744 −7.795 1.00 42.52 C ANISOU 345 CB VAL A 279 5413 5380 5362 33 −38 −9 C ATOM 346 CG1 VAL A 279 −2.993 18.320 −8.244 1.00 41.55 C ANISOU 346 CG1 VAL A 279 5177 5298 5311 13 −94 26 C ATOM 347 CG2 VAL A 279 −1.824 19.896 −6.731 1.00 42.94 C ANISOU 347 CG2 VAL A 279 5406 5427 5483 79 6 17 C ATOM 348 C VAL A 279 −3.633 20.546 −10.115 1.00 43.47 C ANISOU 348 C VAL A 279 5510 5501 5503 −5 −32 −11 C ATOM 349 O VAL A 279 −4.840 20.532 −9.876 1.00 43.83 O ANISOU 349 O VAL A 279 5537 5526 5589 −14 −44 −13 O ATOM 350 N ASP A 280 −3.133 20.472 −11.348 1.00 43.97 N ANISOU 350 N ASP A 280 5575 5537 5591 11 −28 −30 N ATOM 351 CA ASP A 280 −3.963 20.499 −12.557 1.00 44.09 C ANISOU 351 CA ASP A 280 5575 5525 5649 5 −64 −42 C ATOM 352 CB ASP A 280 −4.869 19.252 −12.634 1.00 44.22 C ANISOU 352 CB ASP A 280 5608 5545 5645 17 −30 9 C ATOM 353 CG ASP A 280 −4.151 18.026 −13.197 1.00 43.82 C ANISOU 353 CG ASP A 280 5606 5434 5609 −22 −32 −26 C ATOM 354 OD1 ASP A 280 −3.283 18.156 −14.086 1.00 43.76 O ANISOU 354 OD1 ASP A 280 5538 5390 5698 −27 32 −141 O ATOM 355 OD2 ASP A 280 −4.474 16.915 −12.756 1.00 43.89 O ANISOU 355 OD2 ASP A 280 5597 5469 5609 −7 1 −1 O ATOM 356 C ASP A 280 −4.786 21.799 −12.756 1.00 44.61 C ANISOU 356 C ASP A 280 5619 5577 5751 16 −59 −63 C ATOM 357 O ASP A 280 −5.887 21.761 −13.314 1.00 45.34 O ANISOU 357 O ASP A 280 5661 5665 5897 54 −94 −85 O ATOM 358 N GLY A 281 −4.254 22.941 −12.318 1.00 44.62 N ANISOU 358 N GLY A 281 5615 5564 5774 9 −33 −63 N ATOM 359 CA GLY A 281 −4.920 24.239 −12.522 1.00 43.93 C ANISOU 359 CA GLY A 281 5546 5506 5639 43 −15 −9 C ATOM 360 C GLY A 281 −5.889 24.672 −11.425 1.00 44.05 C ANISOU 360 C GLY A 281 5544 5513 5679 30 −12 3 C ATOM 361 O GLY A 281 −6.548 25.727 −11.535 1.00 44.55 O ANISOU 361 O GLY A 281 5577 5596 5754 17 20 −60 O ATOM 362 N VAL A 282 −5.968 23.871 −10.367 1.00 43.31 N ANISOU 362 N VAL A 282 5472 5421 5561 14 −8 19 N ATOM 363 CA VAL A 282 −6.851 24.121 −9.241 1.00 43.04 C ANISOU 363 CA VAL A 282 5464 5380 5508 26 −25 −11 C ATOM 364 CB VAL A 282 −7.691 22.860 −8.916 1.00 43.44 C ANISOU 364 CB VAL A 282 5511 5459 5536 37 4 −16 C ATOM 365 CG1 VAL A 282 −8.661 23.101 −7.753 1.00 44.12 C ANISOU 365 CG1 VAL A 282 5583 5557 5621 14 21 −60 C ATOM 366 CG2 VAL A 282 −8.442 22.350 −10.169 1.00 42.54 C ANISOU 366 CG2 VAL A 282 5348 5334 5481 10 −71 16 C ATOM 367 C VAL A 282 −5.948 24.480 −8.073 1.00 43.28 C ANISOU 367 C VAL A 282 5520 5394 5528 31 −9 0 C ATOM 368 O VAL A 282 −4.938 23.807 −7.830 1.00 43.32 O ANISOU 368 O VAL A 282 5586 5392 5480 −4 −7 0 O ATOM 369 N GLU A 283 −6.269 25.552 −7.361 1.00 43.39 N ANISOU 369 N GLU A 283 5533 5428 5524 46 −30 −15 N ATOM 370 CA GLU A 283 −5.332 26.039 −6.359 1.00 43.99 C ANISOU 370 CA GLU A 283 5588 5513 5614 39 −27 −44 C ATOM 371 CB GLU A 283 −5.541 27.527 −6.015 1.00 44.14 C ANISOU 371 CB GLU A 283 5616 5518 5637 22 −32 −77 C ATOM 372 CG GLU A 283 −4.233 28.237 −5.566 1.00 45.14 C ANISOU 372 CG GLU A 283 5698 5626 5824 1 −30 −88 C ATOM 373 CD GLU A 283 −4.371 29.755 −5.272 1.00 45.52 C ANISOU 373 CD GLU A 283 5808 5640 5847 63 −22 −79 C ATOM 374 OE1 GLU A 283 −5.169 30.449 −5.936 1.00 45.98 O ANISOU 374 OE1 GLU A 283 5903 5748 5817 168 −129 −128 O ATOM 375 OE2 GLU A 283 −3.643 30.256 −4.381 1.00 45.87 O ANISOU 375 OE2 GLU A 283 5852 5692 5885 80 −99 −213 O ATOM 376 C GLU A 283 −5.379 25.129 −5.137 1.00 43.97 C ANISOU 376 C GLU A 283 5562 5548 5597 37 −9 −38 C ATOM 377 O GLU A 283 −6.407 24.499 −4.844 1.00 44.59 O ANISOU 377 O GLU A 283 5634 5595 5712 35 51 −49 O ATOM 378 N VAL A 284 −4.236 24.998 −4.479 1.00 43.75 N ANISOU 378 N VAL A 284 5529 5540 5552 44 −36 −57 N ATOM 379 CA VAL A 284 −4.120 24.176 −3.273 1.00 43.31 C ANISOU 379 CA VAL A 284 5511 5464 5480 32 −24 −78 C ATOM 380 CB VAL A 284 −3.476 22.788 −3.586 1.00 43.18 C ANISOU 380 CB VAL A 284 5470 5449 5488 12 −27 −98 C ATOM 381 CG1 VAL A 284 −4.052 22.210 −4.898 1.00 43.69 C ANISOU 381 CG1 VAL A 284 5545 5529 5525 52 −3 −77 C ATOM 382 CG2 VAL A 284 −1.955 22.880 −3.689 1.00 42.77 C ANISOU 382 CG2 VAL A 284 5474 5424 5350 28 −8 −121 C ATOM 383 C VAL A 284 −3.295 25.000 −2.284 1.00 43.29 C ANISOU 383 C VAL A 284 5540 5456 5451 58 2 −99 C ATOM 384 O VAL A 284 −2.599 25.934 −2.685 1.00 43.04 O ANISOU 384 O VAL A 284 5517 5406 5431 65 12 −172 O ATOM 385 N HIS A 285 −3.359 24.658 −1.005 1.00 43.83 N ANISOU 385 N HIS A 285 5611 5536 5504 66 −17 −98 N ATOM 386 CA HIS A 285 −2.861 25.567 0.027 1.00 44.27 C ANISOU 386 CA HIS A 285 5668 5587 5565 87 −30 −55 C ATOM 387 CB HIS A 285 −4.050 26.282 0.698 1.00 44.73 C ANISOU 387 CB HIS A 285 5756 5646 5593 75 −33 −36 C ATOM 388 CG HIS A 285 −4.976 26.958 −0.275 1.00 44.92 C ANISOU 388 CG HIS A 285 5649 5723 5694 77 −25 −31 C ATOM 389 ND1 HIS A 285 −4.656 28.136 −0.918 1.00 44.58 N ANISOU 389 ND1 HIS A 285 5612 5609 5716 66 −65 −20 N ATOM 390 CE1 HIS A 285 −5.646 28.483 −1.724 1.00 45.27 C ANISOU 390 CE1 HIS A 285 5786 5744 5667 95 17 −124 C ATOM 391 NE2 HIS A 285 −6.598 27.573 −1.625 1.00 43.43 N ANISOU 391 NE2 HIS A 285 5469 5597 5434 28 51 −17 N ATOM 392 CD2 HIS A 285 −6.199 26.603 −0.735 1.00 44.79 C ANISOU 392 CD2 HIS A 285 5612 5701 5703 19 85 −129 C ATOM 393 C HIS A 285 −1.950 24.866 1.049 1.00 44.64 C ANISOU 393 C HIS A 285 5770 5605 5584 68 −24 −62 C ATOM 394 O HIS A 285 −1.415 25.503 1.966 1.00 44.57 O ANISOU 394 O HIS A 285 5789 5585 5561 88 −2 −76 O ATOM 395 N ASN A 286 −1.729 23.565 0.843 1.00 44.50 N ANISOU 395 N ASN A 286 5789 5535 5584 80 −9 −57 N ATOM 396 CA ASN A 286 −0.962 22.747 1.781 1.00 44.26 C ANISOU 396 CA ASN A 286 5730 5533 5553 67 −12 −83 C ATOM 397 CB ASN A 286 −1.450 21.295 1.735 1.00 44.27 C ANISOU 397 CB ASN A 286 5748 5527 5545 38 6 −34 C ATOM 398 CG ASN A 286 −1.216 20.635 0.373 1.00 44.25 C ANISOU 398 CG ASN A 286 5821 5469 5519 47 −24 −56 C ATOM 399 OD1 ASN A 286 −1.410 21.255 −0.676 1.00 43.91 O ANISOU 399 OD1 ASN A 286 5808 5508 5364 5 −68 −229 O ATOM 400 ND2 ASN A 286 −0.803 19.376 0.391 1.00 44.62 N ANISOU 400 ND2 ASN A 286 5770 5592 5590 125 0 −53 N ATOM 401 C ASN A 286 0.568 22.816 1.625 1.00 44.26 C ANISOU 401 C ASN A 286 5738 5529 5547 68 −18 −77 C ATOM 402 O ASN A 286 1.286 22.107 2.322 1.00 45.01 O ANISOU 402 O ASN A 286 5800 5597 5702 141 −25 −4 O ATOM 403 N ALA A 287 1.077 23.681 0.750 1.00 43.66 N ANISOU 403 N ALA A 287 5666 5502 5421 51 −13 −117 N ATOM 404 CA ALA A 287 2.530 23.836 0.631 1.00 43.65 C ANISOU 404 CA ALA A 287 5588 5531 5464 78 −35 −99 C ATOM 405 CB ALA A 287 2.881 24.633 −0.594 1.00 43.62 C ANISOU 405 CB ALA A 287 5586 5509 5477 102 −29 −107 C ATOM 406 C ALA A 287 3.178 24.463 1.879 1.00 43.97 C ANISOU 406 C ALA A 287 5604 5597 5503 92 −43 −106 C ATOM 407 O ALA A 287 2.539 25.238 2.598 1.00 43.85 O ANISOU 407 O ALA A 287 5569 5635 5454 105 −67 −115 O ATOM 408 N LYS A 288 4.453 24.128 2.104 1.00 44.17 N ANISOU 408 N LYS A 288 5627 5595 5558 72 −23 −103 N ATOM 409 CA LYS A 288 5.217 24.563 3.272 1.00 44.18 C ANISOU 409 CA LYS A 288 5625 5588 5572 29 −45 −99 C ATOM 410 CB LYS A 288 5.689 23.352 4.080 1.00 44.81 C ANISOU 410 CB LYS A 288 5699 5701 5626 40 −42 −150 C ATOM 411 CG LYS A 288 4.593 22.555 4.792 1.00 46.48 C ANISOU 411 CG LYS A 288 5865 5903 5892 −76 11 −29 C ATOM 412 CD LYS A 288 4.200 23.232 6.110 1.00 49.09 C ANISOU 412 CD LYS A 288 6127 6301 6221 −126 134 −75 C ATOM 413 CE LYS A 288 2.938 22.633 6.718 1.00 52.04 C ANISOU 413 CE LYS A 288 6488 6709 6575 43 −23 −39 C ATOM 414 NZ LYS A 288 1.723 22.869 5.865 1.00 52.37 N ANISOU 414 NZ LYS A 288 6568 6732 6598 74 −80 19 N ATOM 415 C LYS A 288 6.420 25.342 2.794 1.00 44.20 C ANISOU 415 C LYS A 288 5613 5591 5587 58 −50 −120 C ATOM 416 O LYS A 288 7.391 24.752 2.339 1.00 44.44 O ANISOU 416 O LYS A 288 5687 5545 5650 97 −149 −161 O ATOM 417 N THR A 289 6.354 26.670 2.864 1.00 44.16 N ANISOU 417 N THR A 289 5601 5569 5607 51 −24 −106 N ATOM 418 CA THR A 289 7.461 27.508 2.418 1.00 44.15 C ANISOU 418 CA THR A 289 5612 5539 5620 39 5 −85 C ATOM 419 CB THR A 289 6.970 28.884 1.928 1.00 44.07 C ANISOU 419 CB THR A 289 5583 5531 5628 43 0 −85 C ATOM 420 OG1 THR A 289 5.785 28.708 1.148 1.00 44.05 O ANISOU 420 OG1 THR A 289 5591 5462 5685 41 30 −182 O ATOM 421 CG2 THR A 289 8.017 29.571 1.057 1.00 43.05 C ANISOU 421 CG2 THR A 289 5481 5379 5495 32 70 −140 C ATOM 422 C THR A 289 8.492 27.654 3.538 1.00 44.68 C ANISOU 422 C THR A 289 5698 5604 5673 42 1 −63 C ATOM 423 O THR A 289 8.134 27.775 4.699 1.00 44.31 O ANISOU 423 O THR A 289 5702 5521 5612 66 10 −51 O ATOM 424 N LYS A 290 9.774 27.609 3.180 1.00 45.81 N ANISOU 424 N LYS A 290 5825 5733 5846 14 −29 −52 N ATOM 425 CA LYS A 290 10.851 27.684 4.165 1.00 46.81 C ANISOU 425 CA LYS A 290 5945 5856 5982 13 −25 −36 C ATOM 426 CB LYS A 290 12.115 26.942 3.676 1.00 47.41 C ANISOU 426 CB LYS A 290 6028 5957 6026 13 4 −53 C ATOM 427 CG LYS A 290 11.847 25.624 2.921 1.00 48.57 C ANISOU 427 CG LYS A 290 6220 6021 6211 −2 67 −25 C ATOM 428 CD LYS A 290 11.104 24.615 3.765 1.00 51.96 C ANISOU 428 CD LYS A 290 6717 6409 6614 73 185 3 C ATOM 429 CE LYS A 290 9.953 24.034 2.979 1.00 49.11 C ANISOU 429 CE LYS A 290 6138 6223 6296 −248 15 22 C ATOM 430 NZ LYS A 290 9.085 23.115 3.801 1.00 53.79 N ANISOU 430 NZ LYS A 290 6802 6907 6729 208 −33 −113 N ATOM 431 C LYS A 290 11.172 29.147 4.440 1.00 46.84 C ANISOU 431 C LYS A 290 5963 5877 5957 0 −26 −24 C ATOM 432 O LYS A 290 11.011 29.996 3.549 1.00 47.40 O ANISOU 432 O LYS A 290 5986 5914 6109 0 −16 −3 O ATOM 433 N PRO A 291 11.592 29.457 5.681 1.00 46.94 N ANISOU 433 N PRO A 291 5985 5900 5950 −14 −59 −1 N ATOM 434 CA PRO A 291 12.091 30.804 5.994 1.00 46.84 C ANISOU 434 CA PRO A 291 5974 5891 5931 −18 −52 −2 C ATOM 435 CB PRO A 291 12.554 30.682 7.462 1.00 46.82 C ANISOU 435 CB PRO A 291 5971 5895 5921 −11 −80 −5 C ATOM 436 CG PRO A 291 12.624 29.199 7.753 1.00 46.92 C ANISOU 436 CG PRO A 291 6015 5881 5931 −28 −125 7 C ATOM 437 CD PRO A 291 11.585 28.578 6.870 1.00 47.19 C ANISOU 437 CD PRO A 291 6015 5909 6005 −25 −71 32 C ATOM 438 C PRO A 291 13.261 31.147 5.075 1.00 46.64 C ANISOU 438 C PRO A 291 5925 5886 5910 5 −30 −4 C ATOM 439 O PRO A 291 14.133 30.296 4.863 1.00 47.04 O ANISOU 439 O PRO A 291 5987 5882 6001 31 −44 −33 O ATOM 440 N ARG A 292 13.264 32.364 4.520 1.00 46.46 N ANISOU 440 N ARG A 292 5884 5850 5915 18 −40 −33 N ATOM 441 CA ARG A 292 14.214 32.738 3.462 1.00 46.02 C ANISOU 441 CA ARG A 292 5853 5808 5821 13 −28 −46 C ATOM 442 CB ARG A 292 13.794 34.046 2.798 1.00 46.45 C ANISOU 442 CB ARG A 292 5904 5847 5896 2 −27 −71 C ATOM 443 CG ARG A 292 13.611 35.259 3.741 1.00 47.04 C ANISOU 443 CG ARG A 292 6025 5960 5886 9 −27 −61 C ATOM 444 CD ARG A 292 13.483 36.560 2.938 1.00 47.13 C ANISOU 444 CD ARG A 292 6000 5923 5984 −61 −73 −30 C ATOM 445 NE ARG A 292 12.698 37.551 3.667 1.00 47.83 N ANISOU 445 NE ARG A 292 6097 5602 6472 −52 16 −335 N ATOM 446 CZ ARG A 292 13.127 38.770 4.014 1.00 57.33 C ANISOU 446 CZ ARG A 292 7795 7019 6967 93 −377 229 C ATOM 447 NH1 ARG A 292 14.348 39.183 3.676 1.00 49.57 N ANISOU 447 NH1 ARG A 292 5617 6420 6794 −337 306 19 N ATOM 448 NH2 ARG A 292 12.320 39.589 4.688 1.00 48.78 N ANISOU 448 NH2 ARG A 292 5976 6056 6501 282 328 −326 N ATOM 449 C ARG A 292 15.654 32.829 3.962 1.00 46.46 C ANISOU 449 C ARG A 292 5866 5923 5862 7 −4 −68 C ATOM 450 O ARG A 292 15.881 33.227 5.111 1.00 46.71 O ANISOU 450 O ARG A 292 5920 5996 5831 8 24 −119 O ATOM 451 N GLU A 293 16.617 32.446 3.108 1.00 46.36 N ANISOU 451 N GLU A 293 5854 5928 5831 24 −8 −73 N ATOM 452 CA GLU A 293 18.053 32.436 3.465 1.00 46.15 C ANISOU 452 CA GLU A 293 5799 5867 5867 0 −8 −62 C ATOM 453 CB GLU A 293 18.639 31.019 3.384 1.00 46.12 C ANISOU 453 CB GLU A 293 5824 5830 5866 1 −58 0 C ATOM 454 CG GLU A 293 17.798 29.901 3.963 1.00 47.34 C ANISOU 454 CG GLU A 293 5940 5960 6085 −28 −21 −38 C ATOM 455 CD GLU A 293 18.535 28.556 3.942 1.00 47.14 C ANISOU 455 CD GLU A 293 5934 5957 6020 39 −105 −55 C ATOM 456 OE1 GLU A 293 19.338 28.315 4.865 1.00 49.59 O ANISOU 456 OE1 GLU A 293 6438 6317 6085 42 −160 10 O ATOM 457 OE2 GLU A 293 18.316 27.742 3.006 1.00 48.92 O ANISOU 457 OE2 GLU A 293 6228 6211 6148 −49 −12 −85 O ATOM 458 C GLU A 293 18.833 33.336 2.514 1.00 45.36 C ANISOU 458 C GLU A 293 5761 5721 5752 −18 −34 −35 C ATOM 459 O GLU A 293 18.457 33.457 1.355 1.00 45.82 O ANISOU 459 O GLU A 293 5815 5803 5791 −15 0 −122 O ATOM 460 N GLU A 294 19.926 33.938 2.994 1.00 45.28 N ANISOU 460 N GLU A 294 5733 5714 5756 −18 −2 −11 N ATOM 461 CA GLU A 294 20.712 34.932 2.222 1.00 44.87 C ANISOU 461 CA GLU A 294 5685 5636 5725 −25 −11 −35 C ATOM 462 CB GLU A 294 21.091 36.145 3.107 1.00 44.81 C ANISOU 462 CB GLU A 294 5705 5615 5706 −20 −7 −42 C ATOM 463 CG GLU A 294 21.392 37.439 2.323 1.00 44.23 C ANISOU 463 CG GLU A 294 5601 5551 5653 −69 −25 −17 C ATOM 464 CD GLU A 294 22.111 38.531 3.140 1.00 44.89 C ANISOU 464 CD GLU A 294 5751 5638 5664 −73 2 −26 C ATOM 465 OE1 GLU A 294 22.680 39.462 2.517 1.00 44.05 O ANISOU 465 OE1 GLU A 294 5729 5440 5566 −143 73 −204 O ATOM 466 OE2 GLU A 294 22.116 38.473 4.393 1.00 46.43 O ANISOU 466 OE2 GLU A 294 5939 5975 5726 −261 −49 46 O ATOM 467 C GLU A 294 21.976 34.325 1.586 1.00 44.97 C ANISOU 467 C GLU A 294 5700 5636 5749 −24 −23 −39 C ATOM 468 O GLU A 294 22.738 33.611 2.247 1.00 45.23 O ANISOU 468 O GLU A 294 5722 5641 5823 4 −34 −91 O ATOM 469 N GLN A 295 22.211 34.628 0.315 1.00 45.12 N ANISOU 469 N GLN A 295 5687 5686 5771 −24 0 −36 N ATOM 470 CA GLN A 295 23.288 33.972 −0.424 1.00 45.81 C ANISOU 470 CA GLN A 295 5768 5739 5898 0 −23 −34 C ATOM 471 CB GLN A 295 22.770 33.466 −1.772 1.00 45.40 C ANISOU 471 CB GLN A 295 5746 5699 5802 14 −9 −34 C ATOM 472 CG GLN A 295 21.458 32.654 −1.658 1.00 45.05 C ANISOU 472 CG GLN A 295 5671 5623 5819 25 −48 39 C ATOM 473 CD GLN A 295 21.627 31.319 −0.930 1.00 50.11 C ANISOU 473 CD GLN A 295 6357 6206 6473 −225 −440 −126 C ATOM 474 OE1 GLN A 295 22.456 30.493 −1.319 1.00 46.51 O ANISOU 474 OE1 GLN A 295 5639 5846 6183 244 39 −244 O ATOM 475 NE2 GLN A 295 20.831 31.098 0.119 1.00 44.53 N ANISOU 475 NE2 GLN A 295 5399 5954 5566 −128 161 −70 N ATOM 476 C GLN A 295 24.555 34.838 −0.580 1.00 46.43 C ANISOU 476 C GLN A 295 5854 5802 5984 26 12 5 C ATOM 477 O GLN A 295 24.486 36.076 −0.703 1.00 47.58 O ANISOU 477 O GLN A 295 6015 5902 6161 59 10 −25 O ATOM 478 N TYR A 296 25.714 34.187 −0.561 1.00 46.40 N ANISOU 478 N TYR A 296 5836 5823 5968 44 40 12 N ATOM 479 CA TYR A 296 26.988 34.887 −0.681 1.00 46.22 C ANISOU 479 CA TYR A 296 5850 5807 5904 6 0 −13 C ATOM 480 CB TYR A 296 28.136 33.947 −0.299 1.00 46.54 C ANISOU 480 CB TYR A 296 5879 5805 5997 11 21 −4 C ATOM 481 CG TYR A 296 28.369 33.809 1.199 1.00 46.10 C ANISOU 481 CG TYR A 296 5899 5814 5802 −12 36 13 C ATOM 482 CD1 TYR A 296 27.454 33.149 2.015 1.00 46.85 C ANISOU 482 CD1 TYR A 296 5952 5814 6034 41 56 −47 C ATOM 483 CE1 TYR A 296 27.676 33.022 3.393 1.00 47.39 C ANISOU 483 CE1 TYR A 296 6047 6058 5900 5 −74 8 C ATOM 484 CZ TYR A 296 28.836 33.551 3.951 1.00 46.56 C ANISOU 484 CZ TYR A 296 5965 5924 5799 −82 4 31 C ATOM 485 OH TYR A 296 29.080 33.427 5.309 1.00 47.47 O ANISOU 485 OH TYR A 296 6193 5903 5937 −47 −8 −40 O ATOM 486 CE2 TYR A 296 29.756 34.202 3.145 1.00 47.62 C ANISOU 486 CE2 TYR A 296 5995 5884 6215 36 51 −59 C ATOM 487 CD2 TYR A 296 29.520 34.324 1.786 1.00 45.57 C ANISOU 487 CD2 TYR A 296 5830 5826 5658 6 −68 15 C ATOM 488 C TYR A 296 27.193 35.501 −2.083 1.00 46.49 C ANISOU 488 C TYR A 296 5907 5803 5951 6 −4 3 C ATOM 489 O TYR A 296 28.292 35.440 −2.666 1.00 46.50 O ANISOU 489 O TYR A 296 5897 5779 5989 −14 −14 −11 O ATOM 490 N ASN A 297 26.119 36.064 −2.631 1.00 46.88 N ANISOU 490 N ASN A 297 5965 5855 5991 17 −15 −17 N ATOM 491 CA ASN A 297 26.216 36.949 −3.792 1.00 47.71 C ANISOU 491 CA ASN A 297 6085 5986 6055 26 −38 −12 C ATOM 492 CB ASN A 297 26.142 36.169 −5.117 1.00 48.85 C ANISOU 492 CB ASN A 297 6260 6139 6160 64 0 −11 C ATOM 493 CG ASN A 297 24.947 35.221 −5.195 1.00 50.92 C ANISOU 493 CG ASN A 297 6460 6440 6445 −63 58 −1 C ATOM 494 OD1 ASN A 297 24.216 35.028 −4.220 1.00 52.42 O ANISOU 494 OD1 ASN A 297 6674 6584 6659 87 34 −103 O ATOM 495 ND2 ASN A 297 24.768 34.605 −6.370 1.00 56.08 N ANISOU 495 ND2 ASN A 297 7171 7114 7021 −39 −31 −25 N ATOM 496 C ASN A 297 25.188 38.088 −3.729 1.00 47.37 C ANISOU 496 C ASN A 297 6034 5962 6003 2 −36 −28 C ATOM 497 O ASN A 297 24.879 38.733 −4.748 1.00 47.54 O ANISOU 497 O ASN A 297 6064 5970 6028 −37 −68 −27 O ATOM 498 N SER A 298 24.679 38.332 −2.516 1.00 47.02 N ANISOU 498 N SER A 298 5921 5925 6017 5 −15 −17 N ATOM 499 CA SER A 298 23.797 39.477 −2.220 1.00 46.71 C ANISOU 499 CA SER A 298 5868 5873 6005 −26 −29 0 C ATOM 500 CB SER A 298 24.415 40.801 −2.693 1.00 46.68 C ANISOU 500 CB SER A 298 5861 5857 6015 −11 −30 45 C ATOM 501 OG SER A 298 25.715 40.961 −2.150 1.00 48.13 O ANISOU 501 OG SER A 298 6019 6020 6244 −95 −25 −12 O ATOM 502 C SER A 298 22.395 39.255 −2.792 1.00 46.18 C ANISOU 502 C SER A 298 5828 5812 5906 −33 −31 13 C ATOM 503 O SER A 298 21.852 40.082 −3.545 1.00 46.36 O ANISOU 503 O SER A 298 5846 5850 5918 −31 −40 43 O ATOM 504 N THR A 299 21.810 38.137 −2.365 1.00 45.58 N ANISOU 504 N THR A 299 5776 5728 5815 −47 −20 0 N ATOM 505 CA THR A 299 20.634 37.558 −2.982 1.00 45.03 C ANISOU 505 CA THR A 299 5757 5632 5719 −42 −23 −30 C ATOM 506 CB THR A 299 21.118 36.736 −4.229 1.00 45.29 C ANISOU 506 CB THR A 299 5793 5664 5749 −38 −58 −30 C ATOM 507 OG1 THR A 299 20.498 37.203 −5.433 1.00 46.45 O ANISOU 507 OG1 THR A 299 5934 5743 5972 21 −92 −8 O ATOM 508 CG2 THR A 299 20.931 35.249 −4.066 1.00 45.09 C ANISOU 508 CG2 THR A 299 5820 5677 5634 −42 −57 −34 C ATOM 509 C THR A 299 19.994 36.667 −1.895 1.00 44.73 C ANISOU 509 C THR A 299 5717 5578 5700 −68 −31 −58 C ATOM 510 O THR A 299 20.734 36.055 −1.103 1.00 44.52 O ANISOU 510 O THR A 299 5722 5570 5623 −99 −47 −90 O ATOM 511 N TYR A 300 18.656 36.609 −1.809 1.00 44.50 N ANISOU 511 N TYR A 300 5713 5554 5640 −37 1 −53 N ATOM 512 CA TYR A 300 18.007 35.534 −1.007 1.00 44.78 C ANISOU 512 CA TYR A 300 5706 5632 5674 −53 22 −106 C ATOM 513 CB TYR A 300 16.841 36.048 −0.117 1.00 45.79 C ANISOU 513 CB TYR A 300 5829 5719 5847 −42 30 −116 C ATOM 514 CG TYR A 300 17.195 37.233 0.822 1.00 47.96 C ANISOU 514 CG TYR A 300 6051 6119 6052 4 19 −11 C ATOM 515 CD1 TYR A 300 17.216 38.547 0.337 1.00 46.79 C ANISOU 515 CD1 TYR A 300 6033 5674 6069 −83 −3 −132 C ATOM 516 CE1 TYR A 300 17.531 39.614 1.158 1.00 47.40 C ANISOU 516 CE1 TYR A 300 6116 6018 5876 −35 4 −148 C ATOM 517 CZ TYR A 300 17.834 39.387 2.495 1.00 47.96 C ANISOU 517 CZ TYR A 300 6083 6090 6048 47 −15 45 C ATOM 518 OH TYR A 300 18.148 40.465 3.318 1.00 46.29 O ANISOU 518 OH TYR A 300 6171 5754 5662 −44 −150 −180 O ATOM 519 CE2 TYR A 300 17.804 38.096 3.006 1.00 47.39 C ANISOU 519 CE2 TYR A 300 6071 5825 6109 49 −44 −39 C ATOM 520 CD2 TYR A 300 17.490 37.029 2.170 1.00 46.41 C ANISOU 520 CD2 TYR A 300 5977 5849 5807 −92 −119 −95 C ATOM 521 C TYR A 300 17.585 34.279 −1.817 1.00 44.23 C ANISOU 521 C TYR A 300 5614 5568 5621 −41 28 −39 C ATOM 522 O TYR A 300 17.452 34.291 −3.056 1.00 44.14 O ANISOU 522 O TYR A 300 5567 5606 5598 −57 27 −81 O ATOM 523 N ARG A 301 17.383 33.193 −1.085 1.00 43.82 N ANISOU 523 N ARG A 301 5560 5519 5571 14 −13 −43 N ATOM 524 CA ARG A 301 16.918 31.936 −1.647 1.00 43.57 C ANISOU 524 CA ARG A 301 5505 5506 5542 −3 −7 −33 C ATOM 525 CB ARG A 301 18.004 30.864 −1.516 1.00 43.36 C ANISOU 525 CB ARG A 301 5504 5467 5501 15 −47 −49 C ATOM 526 CG ARG A 301 17.592 29.478 −2.000 1.00 44.07 C ANISOU 526 CG ARG A 301 5519 5525 5699 −32 −46 −8 C ATOM 527 CD ARG A 301 18.816 28.611 −2.243 1.00 44.34 C ANISOU 527 CD ARG A 301 5415 5556 5873 −21 −27 −130 C ATOM 528 NE ARG A 301 18.503 27.319 −2.846 1.00 45.93 N ANISOU 528 NE ARG A 301 5462 6031 5957 106 −48 −104 N ATOM 529 CZ ARG A 301 18.153 26.232 −2.170 1.00 42.24 C ANISOU 529 CZ ARG A 301 5643 5431 4975 −112 61 −40 C ATOM 530 NH1 ARG A 301 18.055 26.272 −0.844 1.00 47.72 N ANISOU 530 NH1 ARG A 301 5737 6114 6280 8 −101 −45 N ATOM 531 NH2 ARG A 301 17.891 25.102 −2.821 1.00 46.14 N ANISOU 531 NH2 ARG A 301 5495 6132 5903 156 108 67 N ATOM 532 C ARG A 301 15.674 31.523 −0.876 1.00 43.64 C ANISOU 532 C ARG A 301 5540 5535 5503 1 −17 −7 C ATOM 533 O ARG A 301 15.705 31.440 0.362 1.00 44.01 O ANISOU 533 O ARG A 301 5675 5632 5413 52 3 7 O ATOM 534 N VAL A 302 14.574 31.295 −1.598 1.00 43.14 N ANISOU 534 N VAL A 302 5467 5424 5499 −21 −5 −6 N ATOM 535 CA VAL A 302 13.342 30.854 −0.956 1.00 42.72 C ANISOU 535 CA VAL A 302 5403 5315 5512 −37 8 −17 C ATOM 536 CB VAL A 302 12.244 31.957 −0.857 1.00 43.01 C ANISOU 536 CB VAL A 302 5439 5350 5551 −47 13 −13 C ATOM 537 CG1 VAL A 302 11.403 31.739 0.404 1.00 42.56 C ANISOU 537 CG1 VAL A 302 5310 5394 5466 −110 −2 −109 C ATOM 538 CG2 VAL A 302 12.860 33.368 −0.830 1.00 43.35 C ANISOU 538 CG2 VAL A 302 5471 5351 5646 −38 32 −2 C ATOM 539 C VAL A 302 12.803 29.606 −1.643 1.00 42.39 C ANISOU 539 C VAL A 302 5389 5308 5407 −33 −5 −28 C ATOM 540 O VAL A 302 12.657 29.560 −2.863 1.00 41.88 O ANISOU 540 O VAL A 302 5326 5264 5320 −2 −39 −30 O ATOM 541 N VAL A 303 12.529 28.603 −0.810 1.00 42.33 N ANISOU 541 N VAL A 303 5387 5251 5445 −64 −2 −41 N ATOM 542 CA VAL A 303 12.107 27.279 −1.230 1.00 42.18 C ANISOU 542 CA VAL A 303 5328 5270 5427 −26 30 −33 C ATOM 543 CB VAL A 303 13.036 26.184 −0.605 1.00 42.24 C ANISOU 543 CB VAL A 303 5310 5283 5454 −22 24 −23 C ATOM 544 CG1 VAL A 303 12.534 24.790 −0.910 1.00 41.59 C ANISOU 544 CG1 VAL A 303 5214 5202 5384 −38 86 −28 C ATOM 545 CG2 VAL A 303 14.501 26.353 −1.073 1.00 42.85 C ANISOU 545 CG2 VAL A 303 5375 5362 5542 −30 59 −55 C ATOM 546 C VAL A 303 10.674 27.041 −0.748 1.00 41.94 C ANISOU 546 C VAL A 303 5300 5258 5377 −12 27 −38 C ATOM 547 O VAL A 303 10.364 27.277 0.424 1.00 42.19 O ANISOU 547 O VAL A 303 5391 5257 5381 11 76 22 O ATOM 548 N SER A 304 9.801 26.596 −1.648 1.00 41.73 N ANISOU 548 N SER A 304 5238 5240 5375 4 30 −44 N ATOM 549 CA SER A 304 8.505 26.065 −1.231 1.00 41.38 C ANISOU 549 CA SER A 304 5196 5202 5323 −26 5 −31 C ATOM 550 CB SER A 304 7.339 26.861 −1.831 1.00 41.38 C ANISOU 550 CB SER A 304 5184 5211 5327 −22 −8 −54 C ATOM 551 OG SER A 304 6.152 26.629 −1.076 1.00 40.98 O ANISOU 551 OG SER A 304 5221 5152 5196 20 16 −154 O ATOM 552 C SER A 304 8.391 24.579 −1.579 1.00 41.13 C ANISOU 552 C SER A 304 5166 5191 5269 −40 12 −60 C ATOM 553 O SER A 304 8.646 24.190 −2.718 1.00 40.85 O ANISOU 553 O SER A 304 5073 5198 5250 −55 −3 −38 O ATOM 554 N VAL A 305 7.992 23.779 −0.583 1.00 41.00 N ANISOU 554 N VAL A 305 5201 5158 5219 −43 −30 −25 N ATOM 555 CA VAL A 305 7.796 22.329 −0.709 1.00 40.98 C ANISOU 555 CA VAL A 305 5182 5183 5204 −45 −18 −47 C ATOM 556 CB VAL A 305 8.559 21.550 0.407 1.00 40.91 C ANISOU 556 CB VAL A 305 5162 5158 5221 −31 −36 −13 C ATOM 557 CG1 VAL A 305 8.340 20.053 0.282 1.00 40.60 C ANISOU 557 CG1 VAL A 305 5114 5164 5145 −82 −121 −82 C ATOM 558 CG2 VAL A 305 10.044 21.845 0.363 1.00 41.19 C ANISOU 558 CG2 VAL A 305 5200 5121 5326 −85 −25 −36 C ATOM 559 C VAL A 305 6.306 21.921 −0.660 1.00 41.10 C ANISOU 559 C VAL A 305 5186 5211 5215 −36 −14 −73 C ATOM 560 O VAL A 305 5.573 22.260 0.293 1.00 40.73 O ANISOU 560 O VAL A 305 5174 5103 5197 −37 9 −126 O ATOM 561 N LEU A 306 5.887 21.153 −1.666 1.00 41.07 N ANISOU 561 N LEU A 306 5191 5259 5153 −10 −16 −73 N ATOM 562 CA LEU A 306 4.535 20.592 −1.719 1.00 40.99 C ANISOU 562 CA LEU A 306 5188 5219 5166 −16 −40 −36 C ATOM 563 CB LEU A 306 3.748 21.148 −2.918 1.00 41.06 C ANISOU 563 CB LEU A 306 5204 5163 5234 −12 −54 −29 C ATOM 564 CG LEU A 306 2.268 20.728 −3.008 1.00 40.75 C ANISOU 564 CG LEU A 306 5172 5155 5154 53 −5 −10 C ATOM 565 CD1 LEU A 306 1.434 21.455 −1.976 1.00 40.43 C ANISOU 565 CD1 LEU A 306 5289 5183 4888 −31 −17 50 C ATOM 566 CD2 LEU A 306 1.705 20.962 −4.394 1.00 40.87 C ANISOU 566 CD2 LEU A 306 5213 5177 5135 −17 −41 −45 C ATOM 567 C LEU A 306 4.544 19.069 −1.779 1.00 41.08 C ANISOU 567 C LEU A 306 5177 5234 5197 40 −34 −43 C ATOM 568 O LEU A 306 5.110 18.481 −2.693 1.00 40.95 O ANISOU 568 O LEU A 306 5163 5223 5171 14 −57 −46 O ATOM 569 N THR A 307 3.895 18.438 −0.807 1.00 41.54 N ANISOU 569 N THR A 307 5217 5276 5288 40 −35 −55 N ATOM 570 CA THR A 307 3.718 17.004 −0.828 1.00 42.10 C ANISOU 570 CA THR A 307 5268 5362 5363 50 −25 −59 C ATOM 571 CB THR A 307 3.238 16.490 0.534 1.00 42.33 C ANISOU 571 CB THR A 307 5295 5394 5391 46 −26 −62 C ATOM 572 OG1 THR A 307 4.261 16.717 1.510 1.00 43.20 O ANISOU 572 OG1 THR A 307 5387 5546 5479 27 −27 −143 O ATOM 573 CG2 THR A 307 2.943 15.007 0.486 1.00 42.37 C ANISOU 573 CG2 THR A 307 5388 5344 5365 25 −24 −57 C ATOM 574 C THR A 307 2.738 16.624 −1.946 1.00 42.61 C ANISOU 574 C THR A 307 5343 5394 5449 60 −18 −56 C ATOM 575 O THR A 307 1.695 17.279 −2.138 1.00 43.31 O ANISOU 575 O THR A 307 5405 5457 5593 122 −38 −50 O ATOM 576 N VAL A 308 3.092 15.590 −2.705 1.00 41.88 N ANISOU 576 N VAL A 308 5290 5285 5337 54 −5 −77 N ATOM 577 CA VAL A 308 2.175 15.039 −3.688 1.00 41.32 C ANISOU 577 CA VAL A 308 5239 5211 5250 40 −12 −42 C ATOM 578 CB VAL A 308 2.772 15.033 −5.136 1.00 41.12 C ANISOU 578 CB VAL A 308 5250 5192 5180 37 −40 −41 C ATOM 579 CG1 VAL A 308 3.194 16.439 −5.532 1.00 40.83 C ANISOU 579 CG1 VAL A 308 5238 5210 5064 −11 −14 −71 C ATOM 580 CG2 VAL A 308 3.956 14.098 −5.273 1.00 40.49 C ANISOU 580 CG2 VAL A 308 5160 5093 5131 11 −79 −30 C ATOM 581 C VAL A 308 1.678 13.663 −3.230 1.00 41.17 C ANISOU 581 C VAL A 308 5209 5209 5223 41 −23 −66 C ATOM 582 O VAL A 308 2.346 12.959 −2.475 1.00 40.78 O ANISOU 582 O VAL A 308 5262 5123 5107 24 −11 −56 O ATOM 583 N LEU A 309 0.480 13.302 −3.665 1.00 40.99 N ANISOU 583 N LEU A 309 5170 5164 5238 33 −1 −65 N ATOM 584 CA LEU A 309 −0.019 11.972 −3.414 1.00 40.25 C ANISOU 584 CA LEU A 309 5082 5101 5109 31 −12 −14 C ATOM 585 CB LEU A 309 −1.545 11.939 −3.388 1.00 40.44 C ANISOU 585 CB LEU A 309 5114 5151 5098 6 −34 −12 C ATOM 586 CG LEU A 309 −2.214 12.728 −2.261 1.00 41.27 C ANISOU 586 CG LEU A 309 5230 5236 5213 32 −21 0 C ATOM 587 CD1 LEU A 309 −3.692 12.898 −2.547 1.00 42.21 C ANISOU 587 CD1 LEU A 309 5289 5393 5355 41 −69 23 C ATOM 588 CD2 LEU A 309 −2.011 12.051 −0.896 1.00 42.35 C ANISOU 588 CD2 LEU A 309 5357 5365 5366 17 −14 3 C ATOM 589 C LEU A 309 0.561 10.988 −4.429 1.00 39.55 C ANISOU 589 C LEU A 309 4964 5033 5027 −2 −36 −27 C ATOM 590 O LEU A 309 0.800 11.310 −5.591 1.00 38.60 O ANISOU 590 O LEU A 309 4763 4962 4938 33 −16 −94 O ATOM 591 N HIS A 310 0.816 9.795 −3.922 1.00 39.07 N ANISOU 591 N HIS A 310 4906 4954 4982 4 −10 −70 N ATOM 592 CA HIS A 310 1.381 8.692 −4.663 1.00 38.71 C ANISOU 592 CA HIS A 310 4886 4959 4862 −8 4 −35 C ATOM 593 CB HIS A 310 1.371 7.441 −3.778 1.00 37.81 C ANISOU 593 CB HIS A 310 4801 4808 4757 22 −8 −14 C ATOM 594 CG HIS A 310 1.655 6.180 −4.519 1.00 36.41 C ANISOU 594 CG HIS A 310 4652 4701 4480 −32 −3 59 C ATOM 595 ND1 HIS A 310 2.932 5.690 −4.685 1.00 34.00 N ANISOU 595 ND1 HIS A 310 4396 4421 4101 −33 −38 −4 N ATOM 596 CE1 HIS A 310 2.880 4.568 −5.376 1.00 34.94 C ANISOU 596 CE1 HIS A 310 4527 4316 4434 −5 −4 140 C ATOM 597 NE2 HIS A 310 1.614 4.314 −5.667 1.00 34.85 N ANISOU 597 NE2 HIS A 310 4517 4245 4480 61 16 113 N ATOM 598 CD2 HIS A 310 0.829 5.307 −5.140 1.00 35.34 C ANISOU 598 CD2 HIS A 310 4432 4621 4373 9 62 95 C ATOM 599 C HIS A 310 0.644 8.427 −5.973 1.00 38.70 C ANISOU 599 C HIS A 310 4902 4960 4840 −15 −27 −46 C ATOM 600 O HIS A 310 1.277 8.305 −7.027 1.00 37.82 O ANISOU 600 O HIS A 310 4807 4841 4722 −36 −23 −44 O ATOM 601 N GLN A 311 −0.684 8.335 −5.883 1.00 39.22 N ANISOU 601 N GLN A 311 5005 5002 4894 −3 19 −72 N ATOM 602 CA GLN A 311 −1.541 8.052 −7.037 1.00 39.83 C ANISOU 602 CA GLN A 311 5068 5069 4997 4 −14 −53 C ATOM 603 CB GLN A 311 −2.941 7.575 −6.615 1.00 39.64 C ANISOU 603 CB GLN A 311 5050 5101 4910 8 −4 −67 C ATOM 604 CG GLN A 311 −3.814 8.592 −5.859 1.00 41.81 C ANISOU 604 CG GLN A 311 5349 5334 5203 34 4 3 C ATOM 605 CD GLN A 311 −3.637 8.590 −4.319 1.00 44.78 C ANISOU 605 CD GLN A 311 5943 5685 5387 −47 37 −57 C ATOM 606 OE1 GLN A 311 −2.590 8.195 −3.771 1.00 43.08 O ANISOU 606 OE1 GLN A 311 5380 5867 5120 97 −37 −211 O ATOM 607 NE2 GLN A 311 −4.681 9.036 −3.622 1.00 43.39 N ANISOU 607 NE2 GLN A 311 5563 5667 5256 170 135 −45 N ATOM 608 C GLN A 311 −1.594 9.229 −7.995 1.00 39.90 C ANISOU 608 C GLN A 311 5049 5064 5045 −16 −7 −58 C ATOM 609 O GLN A 311 −1.555 9.046 −9.229 1.00 40.38 O ANISOU 609 O GLN A 311 5051 5083 5207 −72 45 −44 O ATOM 610 N ASP A 312 −1.642 10.431 −7.425 1.00 39.64 N ANISOU 610 N ASP A 312 5014 5048 4998 −22 −10 −70 N ATOM 611 CA ASP A 312 −1.723 11.662 −8.196 1.00 39.61 C ANISOU 611 CA ASP A 312 5003 5033 5013 −9 −1 −80 C ATOM 612 CB ASP A 312 −1.841 12.891 −7.276 1.00 39.79 C ANISOU 612 CB ASP A 312 5039 5090 4987 −8 −26 −109 C ATOM 613 CG ASP A 312 −3.202 13.029 −6.639 1.00 40.70 C ANISOU 613 CG ASP A 312 5104 5172 5186 −39 18 −131 C ATOM 614 OD1 ASP A 312 −4.081 12.167 −6.863 1.00 42.10 O ANISOU 614 OD1 ASP A 312 5286 5405 5303 51 91 −230 O ATOM 615 OD2 ASP A 312 −3.389 14.010 −5.888 1.00 43.00 O ANISOU 615 OD2 ASP A 312 5297 5347 5692 −20 69 1 O ATOM 616 C ASP A 312 −0.518 11.833 −9.091 1.00 38.73 C ANISOU 616 C ASP A 312 4902 4899 4914 11 12 −121 C ATOM 617 O ASP A 312 −0.655 12.241 −10.251 1.00 39.28 O ANISOU 617 O ASP A 312 4946 4963 5014 −3 85 −129 O ATOM 618 N TRP A 313 0.656 11.537 −8.543 1.00 37.61 N ANISOU 618 N TRP A 313 4772 4760 4758 −8 10 −110 N ATOM 619 CA TRP A 313 1.907 11.678 −9.279 1.00 36.85 C ANISOU 619 CA TRP A 313 4619 4730 4649 −7 −60 −88 C ATOM 620 CB TRP A 313 3.129 11.487 −8.357 1.00 36.13 C ANISOU 620 CB TRP A 313 4550 4680 4494 −8 −75 −20 C ATOM 621 CG TRP A 313 4.441 11.751 −9.093 1.00 35.34 C ANISOU 621 CG TRP A 313 4466 4697 4264 43 −176 −103 C ATOM 622 CD1 TRP A 313 5.343 10.827 −9.512 1.00 35.07 C ANISOU 622 CD1 TRP A 313 4501 4715 4109 −17 −197 −29 C ATOM 623 NE1 TRP A 313 6.406 11.442 −10.135 1.00 34.18 N ANISOU 623 NE1 TRP A 313 4463 4332 4191 −11 −148 20 N ATOM 624 CE2 TRP A 313 6.187 12.796 −10.146 1.00 35.05 C ANISOU 624 CE2 TRP A 313 4446 4707 4162 −75 −196 −97 C ATOM 625 CD2 TRP A 313 4.954 13.028 −9.497 1.00 33.56 C ANISOU 625 CD2 TRP A 313 4353 4503 3893 −30 −139 4 C ATOM 626 CE3 TRP A 313 4.501 14.348 −9.354 1.00 32.76 C ANISOU 626 CE3 TRP A 313 4229 4481 3734 0 −202 82 C ATOM 627 CZ3 TRP A 313 5.279 15.377 −9.862 1.00 35.07 C ANISOU 627 CZ3 TRP A 313 4469 4665 4192 −3 −238 41 C ATOM 628 CH2 TRP A 313 6.493 15.113 −10.532 1.00 34.15 C ANISOU 628 CH2 TRP A 313 4324 4418 4233 −22 −142 86 C ATOM 629 CZ2 TRP A 313 6.959 13.833 −10.685 1.00 35.30 C ANISOU 629 CZ2 TRP A 313 4442 4682 4288 −11 −136 −64 C ATOM 630 C TRP A 313 1.998 10.709 −10.457 1.00 36.14 C ANISOU 630 C TRP A 313 4477 4717 4537 0 −78 −55 C ATOM 631 O TRP A 313 2.313 11.107 −11.562 1.00 35.50 O ANISOU 631 O TRP A 313 4328 4708 4450 45 −104 −91 O ATOM 632 N LEU A 314 1.765 9.438 −10.157 1.00 35.58 N ANISOU 632 N LEU A 314 4401 4613 4503 −13 −76 −52 N ATOM 633 CA LEU A 314 1.729 8.348 −11.105 1.00 35.56 C ANISOU 633 CA LEU A 314 4445 4567 4497 28 5 −28 C ATOM 634 CB LEU A 314 1.574 7.016 −10.361 1.00 35.07 C ANISOU 634 CB LEU A 314 4405 4507 4410 −12 18 −47 C ATOM 635 CG LEU A 314 2.750 6.390 −9.621 1.00 34.91 C ANISOU 635 CG LEU A 314 4444 4487 4331 −14 −28 −14 C ATOM 636 CD1 LEU A 314 2.290 5.110 −8.980 1.00 35.56 C ANISOU 636 CD1 LEU A 314 4432 4609 4471 17 −104 −47 C ATOM 637 CD2 LEU A 314 3.922 6.091 −10.522 1.00 34.75 C ANISOU 637 CD2 LEU A 314 4491 4405 4306 41 −118 −7 C ATOM 638 C LEU A 314 0.618 8.461 −12.146 1.00 35.53 C ANISOU 638 C LEU A 314 4500 4504 4495 −33 −16 −32 C ATOM 639 O LEU A 314 0.782 7.941 −13.236 1.00 35.75 O ANISOU 639 O LEU A 314 4455 4563 4564 −36 −3 17 O ATOM 640 N ASN A 315 −0.504 9.110 −11.802 1.00 35.21 N ANISOU 640 N ASN A 315 4478 4419 4481 −76 −52 −36 N ATOM 641 CA ASN A 315 −1.580 9.402 −12.763 1.00 35.05 C ANISOU 641 CA ASN A 315 4531 4373 4411 −33 2 −57 C ATOM 642 CB ASN A 315 −2.958 9.466 −12.074 1.00 35.00 C ANISOU 642 CB ASN A 315 4513 4349 4433 −40 −16 −16 C ATOM 643 CG ASN A 315 −3.500 8.086 −11.725 1.00 35.48 C ANISOU 643 CG ASN A 315 4451 4447 4582 −119 −42 −126 C ATOM 644 OD1 ASN A 315 −3.252 7.128 −12.445 1.00 33.89 O ANISOU 644 OD1 ASN A 315 4226 4285 4363 −276 −266 −225 O ATOM 645 ND2 ASN A 315 −4.209 7.977 −10.601 1.00 34.22 N ANISOU 645 ND2 ASN A 315 4214 4375 4410 −223 34 −77 N ATOM 646 C ASN A 315 −1.379 10.647 −13.620 1.00 34.87 C ANISOU 646 C ASN A 315 4516 4388 4343 −23 1 −67 C ATOM 647 O ASN A 315 −2.321 11.112 −14.237 1.00 34.63 O ANISOU 647 O ASN A 315 4549 4319 4289 −30 41 −65 O ATOM 648 N GLY A 316 −0.171 11.197 −13.639 1.00 35.22 N ANISOU 648 N GLY A 316 4517 4502 4360 6 3 −37 N ATOM 649 CA GLY A 316 0.165 12.331 −14.516 1.00 35.71 C ANISOU 649 CA GLY A 316 4494 4573 4500 −5 −1 −46 C ATOM 650 C GLY A 316 −0.397 13.709 −14.206 1.00 35.78 C ANISOU 650 C GLY A 316 4467 4612 4516 −28 −42 −50 C ATOM 651 O GLY A 316 −0.366 14.597 −15.058 1.00 35.56 O ANISOU 651 O GLY A 316 4332 4654 4522 −7 −66 −97 O ATOM 652 N LYS A 317 −0.902 13.890 −12.991 1.00 36.31 N ANISOU 652 N LYS A 317 4528 4698 4570 −30 −42 −59 N ATOM 653 CA LYS A 317 −1.315 15.203 −12.493 1.00 37.07 C ANISOU 653 CA LYS A 317 4699 4780 4606 −7 −28 −47 C ATOM 654 CB LYS A 317 −1.755 15.110 −11.031 1.00 37.28 C ANISOU 654 CB LYS A 317 4696 4803 4663 −25 −9 −80 C ATOM 655 CG LYS A 317 −3.033 14.325 −10.813 1.00 38.63 C ANISOU 655 CG LYS A 317 4886 4905 4884 −49 −2 −35 C ATOM 656 CD LYS A 317 −3.784 14.865 −9.616 1.00 39.92 C ANISOU 656 CD LYS A 317 4984 5172 5010 19 65 −11 C ATOM 657 CE LYS A 317 −5.138 14.188 −9.434 1.00 40.77 C ANISOU 657 CE LYS A 317 5161 5227 5100 116 79 −35 C ATOM 658 NZ LYS A 317 −6.015 15.042 −8.572 1.00 41.78 N ANISOU 658 NZ LYS A 317 5263 5525 5086 124 26 96 N ATOM 659 C LYS A 317 −0.180 16.214 −12.629 1.00 37.17 C ANISOU 659 C LYS A 317 4747 4823 4551 −27 −28 −80 C ATOM 660 O LYS A 317 0.980 15.859 −12.440 1.00 37.06 O ANISOU 660 O LYS A 317 4756 4804 4520 −34 84 −155 O ATOM 661 N GLU A 318 −0.525 17.462 −12.941 1.00 37.60 N ANISOU 661 N GLU A 318 4848 4817 4621 −12 −30 −99 N ATOM 662 CA GLU A 318 0.452 18.519 −13.238 1.00 40.15 C ANISOU 662 CA GLU A 318 5164 5032 5058 −54 −30 −117 C ATOM 663 CB GLU A 318 0.052 19.263 −14.523 1.00 39.10 C ANISOU 663 CB GLU A 318 5034 4963 4859 −60 −36 −45 C ATOM 664 CG GLU A 318 0.271 18.492 −15.799 1.00 41.10 C ANISOU 664 CG GLU A 318 5181 5159 5275 −19 9 −184 C ATOM 665 CD GLU A 318 0.118 19.357 −17.031 1.00 38.29 C ANISOU 665 CD GLU A 318 4479 5157 4911 107 25 128 C ATOM 666 OE1 GLU A 318 1.126 19.619 −17.700 1.00 46.87 O ANISOU 666 OE1 GLU A 318 6088 5866 5854 −27 −89 −299 O ATOM 667 OE2 GLU A 318 −0.997 19.815 −17.354 1.00 46.71 O ANISOU 667 OE2 GLU A 318 6297 5852 5598 −240 197 −211 O ATOM 668 C GLU A 318 0.583 19.530 −12.092 1.00 38.57 C ANISOU 668 C GLU A 318 4938 4896 4818 −31 −53 −85 C ATOM 669 O GLU A 318 −0.425 20.012 −11.578 1.00 38.13 O ANISOU 669 O GLU A 318 4923 4789 4774 −39 −135 −122 O ATOM 670 N TYR A 319 1.821 19.851 −11.705 1.00 38.78 N ANISOU 670 N TYR A 319 4998 4868 4867 −48 −21 −87 N ATOM 671 CA TYR A 319 2.079 20.701 −10.529 1.00 39.04 C ANISOU 671 CA TYR A 319 5000 4916 4916 −26 −53 −60 C ATOM 672 CB TYR A 319 2.884 19.937 −9.476 1.00 38.84 C ANISOU 672 CB TYR A 319 4972 4905 4879 −26 −72 −93 C ATOM 673 CG TYR A 319 2.172 18.689 −8.973 1.00 37.38 C ANISOU 673 CG TYR A 319 4832 4650 4719 −86 32 −11 C ATOM 674 CD1 TYR A 319 2.258 17.460 −9.673 1.00 38.12 C ANISOU 674 CD1 TYR A 319 4829 4759 4893 7 −6 −24 C ATOM 675 CE1 TYR A 319 1.593 16.320 −9.215 1.00 36.71 C ANISOU 675 CE1 TYR A 319 4646 4520 4781 −18 −157 −183 C ATOM 676 CZ TYR A 319 0.834 16.411 −8.065 1.00 36.25 C ANISOU 676 CZ TYR A 319 4621 4505 4646 −178 −20 −20 C ATOM 677 OH TYR A 319 0.166 15.322 −7.573 1.00 39.25 O ANISOU 677 OH TYR A 319 5143 5009 4759 54 −255 −66 O ATOM 678 CE2 TYR A 319 0.725 17.612 −7.374 1.00 37.40 C ANISOU 678 CE2 TYR A 319 4648 4766 4794 39 −30 −82 C ATOM 679 CD2 TYR A 319 1.389 18.736 −7.836 1.00 36.44 C ANISOU 679 CD2 TYR A 319 4701 4522 4622 68 −116 −138 C ATOM 680 C TYR A 319 2.745 22.028 −10.889 1.00 39.24 C ANISOU 680 C TYR A 319 5014 4957 4938 −40 −57 −97 C ATOM 681 O TYR A 319 3.918 22.071 −11.266 1.00 39.11 O ANISOU 681 O TYR A 319 4977 4930 4951 −22 −85 −124 O ATOM 682 N LYS A 320 1.961 23.101 −10.790 1.00 39.64 N ANISOU 682 N LYS A 320 5077 5012 4970 −1 −53 −107 N ATOM 683 CA LYS A 320 2.398 24.454 −11.109 1.00 40.77 C ANISOU 683 CA LYS A 320 5193 5147 5150 −23 −13 −85 C ATOM 684 CB LYS A 320 1.310 25.173 −11.921 1.00 40.27 C ANISOU 684 CB LYS A 320 5129 5063 5107 21 −64 −45 C ATOM 685 CG LYS A 320 1.704 26.535 −12.547 1.00 46.09 C ANISOU 685 CG LYS A 320 6665 5282 5563 −484 537 −253 C ATOM 686 CD LYS A 320 0.609 27.084 −13.492 1.00 38.31 C ANISOU 686 CD LYS A 320 4594 5097 4864 287 −177 117 C ATOM 687 CE LYS A 320 −0.733 27.374 −12.771 1.00 47.84 C ANISOU 687 CE LYS A 320 6419 5509 6250 −354 338 −151 C ATOM 688 NZ LYS A 320 −1.892 27.744 −13.695 1.00 37.09 N ANISOU 688 NZ LYS A 320 4077 5276 4737 675 −795 197 N ATOM 689 C LYS A 320 2.759 25.250 −9.840 1.00 40.86 C ANISOU 689 C LYS A 320 5229 5123 5173 18 −10 −35 C ATOM 690 O LYS A 320 2.030 25.259 −8.843 1.00 40.72 O ANISOU 690 O LYS A 320 5252 5105 5111 38 −28 −57 O ATOM 691 N CYS A 321 3.915 25.888 −9.894 1.00 41.68 N ANISOU 691 N CYS A 321 5350 5199 5287 18 −5 −32 N ATOM 692 CA CYS A 321 4.373 26.788 −8.860 1.00 42.24 C ANISOU 692 CA CYS A 321 5408 5260 5381 31 1 −18 C ATOM 693 CB CYS A 321 5.763 26.361 −8.379 1.00 42.35 C ANISOU 693 CB CYS A 321 5400 5274 5415 9 −9 −35 C ATOM 694 SG CYS A 321 6.500 27.426 −7.128 1.00 42.58 S ANISOU 694 SG CYS A 321 5484 5270 5421 50 −14 −77 S ATOM 695 C CYS A 321 4.411 28.191 −9.472 1.00 42.81 C ANISOU 695 C CYS A 321 5475 5310 5480 8 16 −9 C ATOM 696 O CYS A 321 5.156 28.422 −10.460 1.00 42.92 O ANISOU 696 O CYS A 321 5461 5316 5528 24 61 −9 O ATOM 697 N LYS A 322 3.570 29.085 −8.924 1.00 42.76 N ANISOU 697 N LYS A 322 5459 5348 5439 17 33 −26 N ATOM 698 CA LYS A 322 3.565 30.511 −9.287 1.00 42.86 C ANISOU 698 CA LYS A 322 5437 5374 5471 56 −15 9 C ATOM 699 CB LYS A 322 2.147 31.078 −9.435 1.00 43.24 C ANISOU 699 CB LYS A 322 5551 5409 5468 25 18 −36 C ATOM 700 CG LYS A 322 2.064 32.254 −10.426 1.00 44.33 C ANISOU 700 CG LYS A 322 5694 5515 5634 14 −35 −25 C ATOM 701 CD LYS A 322 0.634 32.831 −10.569 1.00 45.76 C ANISOU 701 CD LYS A 322 5744 5672 5970 −51 35 −111 C ATOM 702 CE LYS A 322 0.468 33.530 −11.933 1.00 40.52 C ANISOU 702 CE LYS A 322 4781 4752 5862 1225 −190 243 C ATOM 703 NZ LYS A 322 −0.714 34.463 −12.029 1.00 52.37 N ANISOU 703 NZ LYS A 322 7085 6923 5891 −760 97 −81 N ATOM 704 C LYS A 322 4.352 31.332 −8.284 1.00 42.99 C ANISOU 704 C LYS A 322 5456 5381 5497 1 30 0 C ATOM 705 O LYS A 322 4.196 31.206 −7.065 1.00 42.73 O ANISOU 705 O LYS A 322 5391 5361 5482 50 28 −24 O ATOM 706 N VAL A 323 5.220 32.172 −8.823 1.00 43.35 N ANISOU 706 N VAL A 323 5501 5422 5545 −16 −11 12 N ATOM 707 CA VAL A 323 6.125 32.979 −8.025 1.00 42.78 C ANISOU 707 CA VAL A 323 5428 5354 5472 −14 −20 −20 C ATOM 708 CB VAL A 323 7.586 32.528 −8.212 1.00 42.83 C ANISOU 708 CB VAL A 323 5397 5384 5491 −25 −20 12 C ATOM 709 CG1 VAL A 323 8.557 33.587 −7.703 1.00 41.95 C ANISOU 709 CG1 VAL A 323 5327 5304 5305 17 −15 −62 C ATOM 710 CG2 VAL A 323 7.811 31.192 −7.509 1.00 43.44 C ANISOU 710 CG2 VAL A 323 5415 5409 5681 24 −7 −27 C ATOM 711 C VAL A 323 5.942 34.423 −8.432 1.00 42.81 C ANISOU 711 C VAL A 323 5441 5360 5463 −14 −20 −44 C ATOM 712 O VAL A 323 6.102 34.796 −9.621 1.00 42.57 O ANISOU 712 O VAL A 323 5408 5357 5408 −89 29 −68 O ATOM 713 N SER A 324 5.597 35.219 −7.421 1.00 42.64 N ANISOU 713 N SER A 324 5419 5369 5414 −2 −9 −59 N ATOM 714 CA SER A 324 5.207 36.605 −7.584 1.00 42.51 C ANISOU 714 CA SER A 324 5426 5317 5408 −11 −3 −71 C ATOM 715 CB SER A 324 3.750 36.783 −7.154 1.00 42.22 C ANISOU 715 CB SER A 324 5364 5281 5394 16 −26 −72 C ATOM 716 OG SER A 324 2.864 36.502 −8.216 1.00 41.92 O ANISOU 716 OG SER A 324 5332 5206 5387 19 53 −145 O ATOM 717 C SER A 324 6.104 37.522 −6.760 1.00 43.07 C ANISOU 717 C SER A 324 5479 5404 5481 −32 −10 −59 C ATOM 718 O SER A 324 6.352 37.271 −5.575 1.00 42.70 O ANISOU 718 O SER A 324 5452 5352 5420 −41 −68 −72 O ATOM 719 N ASN A 325 6.571 38.596 −7.396 1.00 44.33 N ANISOU 719 N ASN A 325 5633 5581 5628 −43 19 −83 N ATOM 720 CA ASN A 325 7.437 39.570 −6.724 1.00 45.40 C ANISOU 720 CA ASN A 325 5802 5719 5727 −31 13 −62 C ATOM 721 CB ASN A 325 8.886 39.074 −6.668 1.00 45.78 C ANISOU 721 CB ASN A 325 5807 5817 5767 −17 24 −48 C ATOM 722 CG ASN A 325 9.749 39.887 −5.724 1.00 47.87 C ANISOU 722 CG ASN A 325 6012 6087 6088 −56 −15 −90 C ATOM 723 OD1 ASN A 325 9.252 40.493 −4.758 1.00 51.73 O ANISOU 723 OD1 ASN A 325 6606 6613 6436 43 24 −165 O ATOM 724 ND2 ASN A 325 11.055 39.898 −5.986 1.00 49.10 N ANISOU 724 ND2 ASN A 325 6088 6256 6312 27 72 40 N ATOM 725 C ASN A 325 7.379 40.924 −7.396 1.00 45.82 C ANISOU 725 C ASN A 325 5904 5749 5757 −16 29 −60 C ATOM 726 O ASN A 325 7.568 41.032 −8.624 1.00 46.11 O ANISOU 726 O ASN A 325 5966 5817 5735 8 24 −72 O ATOM 727 N LYS A 326 7.106 41.945 −6.581 1.00 46.40 N ANISOU 727 N LYS A 326 5999 5832 5798 6 54 −106 N ATOM 728 CA LYS A 326 7.088 43.359 −6.999 1.00 46.62 C ANISOU 728 CA LYS A 326 6011 5855 5844 −28 75 −54 C ATOM 729 CB LYS A 326 6.596 44.250 −5.847 1.00 46.66 C ANISOU 729 CB LYS A 326 6026 5795 5908 −14 78 −87 C ATOM 730 CG LYS A 326 5.693 43.541 −4.808 1.00 46.45 C ANISOU 730 CG LYS A 326 6008 5829 5811 1 106 −66 C ATOM 731 CD LYS A 326 5.224 44.500 −3.730 1.00 46.76 C ANISOU 731 CD LYS A 326 6052 5871 5842 −23 128 −26 C ATOM 732 CE LYS A 326 4.627 43.755 −2.527 1.00 47.92 C ANISOU 732 CE LYS A 326 6179 6075 5951 −22 57 57 C ATOM 733 NZ LYS A 326 3.645 44.615 −1.795 1.00 46.79 N ANISOU 733 NZ LYS A 326 6086 5950 5742 84 143 63 N ATOM 734 C LYS A 326 8.492 43.774 −7.398 1.00 47.27 C ANISOU 734 C LYS A 326 6099 5947 5913 −66 66 −56 C ATOM 735 O LYS A 326 9.180 44.487 −6.654 1.00 48.18 O ANISOU 735 O LYS A 326 6284 6034 5987 −48 39 −1 O ATOM 736 N ALA A 327 8.916 43.299 −8.570 1.00 48.04 N ANISOU 736 N ALA A 327 6153 6097 6003 −80 76 −72 N ATOM 737 CA ALA A 327 10.266 43.501 −9.120 1.00 48.25 C ANISOU 737 CA ALA A 327 6151 6154 6027 −80 65 −43 C ATOM 738 CB ALA A 327 11.322 42.844 −8.247 1.00 48.36 C ANISOU 738 CB ALA A 327 6156 6153 6064 −60 40 −40 C ATOM 739 C ALA A 327 10.305 42.901 −10.522 1.00 48.66 C ANISOU 739 C ALA A 327 6209 6190 6089 −90 89 −47 C ATOM 740 O ALA A 327 11.017 43.403 −11.396 1.00 49.36 O ANISOU 740 O ALA A 327 6216 6343 6195 −143 160 −32 O ATOM 741 N LEU A 328 9.531 41.834 −10.732 1.00 48.61 N ANISOU 741 N LEU A 328 6214 6128 6125 −66 61 −36 N ATOM 742 CA LEU A 328 9.411 41.216 −12.056 1.00 48.46 C ANISOU 742 CA LEU A 328 6198 6118 6096 −1 44 −5 C ATOM 743 CB LEU A 328 8.993 39.743 −11.934 1.00 48.19 C ANISOU 743 CB LEU A 328 6138 6070 6100 13 48 9 C ATOM 744 CG LEU A 328 10.002 38.667 −11.520 1.00 48.23 C ANISOU 744 CG LEU A 328 6153 6038 6133 9 15 36 C ATOM 745 CD1 LEU A 328 9.283 37.550 −10.802 1.00 46.88 C ANISOU 745 CD1 LEU A 328 5933 5924 5956 −6 2 89 C ATOM 746 CD2 LEU A 328 10.797 38.134 −12.728 1.00 46.84 C ANISOU 746 CD2 LEU A 328 6059 5772 5966 30 −18 8 C ATOM 747 C LEU A 328 8.377 41.962 −12.905 1.00 48.82 C ANISOU 747 C LEU A 328 6215 6170 6161 42 47 3 C ATOM 748 O LEU A 328 7.346 42.391 −12.370 1.00 49.39 O ANISOU 748 O LEU A 328 6288 6214 6264 62 78 −17 O ATOM 749 N PRO A 329 8.627 42.096 −14.233 1.00 48.99 N ANISOU 749 N PRO A 329 6230 6210 6172 52 35 20 N ATOM 750 CA PRO A 329 7.583 42.691 −15.083 1.00 48.98 C ANISOU 750 CA PRO A 329 6183 6202 6224 38 16 11 C ATOM 751 CB PRO A 329 8.121 42.512 −16.512 1.00 48.81 C ANISOU 751 CB PRO A 329 6180 6199 6165 26 5 20 C ATOM 752 CG PRO A 329 9.255 41.473 −16.402 1.00 49.03 C ANISOU 752 CG PRO A 329 6235 6230 6164 50 −11 24 C ATOM 753 CD PRO A 329 9.824 41.712 −15.016 1.00 49.11 C ANISOU 753 CD PRO A 329 6245 6228 6184 54 26 18 C ATOM 754 C PRO A 329 6.275 41.925 −14.898 1.00 49.47 C ANISOU 754 C PRO A 329 6237 6232 6324 32 28 24 C ATOM 755 O PRO A 329 5.199 42.555 −14.828 1.00 50.19 O ANISOU 755 O PRO A 329 6307 6296 6465 82 62 20 O ATOM 756 N ALA A 330 6.386 40.588 −14.815 1.00 49.21 N ANISOU 756 N ALA A 330 6236 6180 6279 31 21 35 N ATOM 757 CA ALA A 330 5.267 39.685 −14.511 1.00 48.97 C ANISOU 757 CA ALA A 330 6221 6162 6220 0 15 46 C ATOM 758 C ALA A 330 5.734 38.538 −13.611 1.00 48.44 C ANISOU 758 C ALA A 330 6145 6082 6176 14 16 18 C ATOM 759 O ALA A 330 6.938 38.338 −13.433 1.00 48.63 O ANISOU 759 O ALA A 330 6183 6095 6196 22 6 21 O ATOM 760 CB ALA A 330 4.638 39.170 −15.796 1.00 49.45 C ANISOU 760 CB ALA A 330 6282 6264 6242 −3 41 22 C ATOM 761 N SER A 331 4.774 37.789 −13.057 1.00 47.86 N ANISOU 761 N SER A 331 6127 5988 6070 15 23 18 N ATOM 762 CA SER A 331 5.044 36.573 −12.279 1.00 47.47 C ANISOU 762 CA SER A 331 6076 5962 5998 20 52 −2 C ATOM 763 CB SER A 331 3.732 35.989 −11.759 1.00 47.76 C ANISOU 763 CB SER A 331 6111 5968 6068 −34 62 −6 C ATOM 764 OG SER A 331 3.328 36.636 −10.566 1.00 49.07 O ANISOU 764 OG SER A 331 6264 6200 6180 −65 112 −82 O ATOM 765 C SER A 331 5.784 35.490 −13.063 1.00 46.93 C ANISOU 765 C SER A 331 6026 5870 5936 45 11 17 C ATOM 766 O SER A 331 5.841 35.530 −14.306 1.00 46.88 O ANISOU 766 O SER A 331 6028 5874 5910 64 −8 49 O ATOM 767 N ILE A 332 6.349 34.524 −12.326 1.00 46.46 N ANISOU 767 N ILE A 332 5955 5859 5838 52 8 10 N ATOM 768 CA ILE A 332 6.997 33.345 −12.921 1.00 45.16 C ANISOU 768 CA ILE A 332 5784 5664 5710 32 1 −50 C ATOM 769 CB ILE A 332 8.447 33.111 −12.418 1.00 45.49 C ANISOU 769 CB ILE A 332 5827 5705 5750 38 30 −55 C ATOM 770 CG1 ILE A 332 9.226 34.425 −12.299 1.00 45.00 C ANISOU 770 CG1 ILE A 332 5746 5597 5752 3 1 −96 C ATOM 771 CD1 ILE A 332 10.603 34.263 −11.676 1.00 44.54 C ANISOU 771 CD1 ILE A 332 5753 5514 5657 62 −2 −95 C ATOM 772 CG2 ILE A 332 9.199 32.107 −13.353 1.00 46.08 C ANISOU 772 CG2 ILE A 332 5899 5694 5914 5 5 −81 C ATOM 773 C ILE A 332 6.212 32.091 −12.614 1.00 45.22 C ANISOU 773 C ILE A 332 5786 5699 5695 11 19 −20 C ATOM 774 O ILE A 332 5.959 31.784 −11.447 1.00 45.15 O ANISOU 774 O ILE A 332 5742 5739 5670 47 −18 −46 O ATOM 775 N GLU A 333 5.846 31.365 −13.675 1.00 45.30 N ANISOU 775 N GLU A 333 5774 5702 5736 −22 17 4 N ATOM 776 CA GLU A 333 5.198 30.059 −13.561 1.00 45.04 C ANISOU 776 CA GLU A 333 5702 5658 5752 6 34 −3 C ATOM 777 CB GLU A 333 3.920 30.002 −14.403 1.00 45.11 C ANISOU 777 CB GLU A 333 5759 5677 5704 −3 34 16 C ATOM 778 CG GLU A 333 2.743 30.808 −13.893 1.00 46.10 C ANISOU 778 CG GLU A 333 5802 5868 5845 9 −6 18 C ATOM 779 CD GLU A 333 1.567 30.734 −14.839 1.00 47.62 C ANISOU 779 CD GLU A 333 6049 5979 6064 −46 8 80 C ATOM 780 OE1 GLU A 333 0.502 31.308 −14.524 1.00 49.66 O ANISOU 780 OE1 GLU A 333 6257 6258 6355 147 141 19 O ATOM 781 OE2 GLU A 333 1.706 30.101 −15.911 1.00 49.76 O ANISOU 781 OE2 GLU A 333 6561 6209 6136 66 28 −65 O ATOM 782 C GLU A 333 6.112 28.961 −14.076 1.00 44.61 C ANISOU 782 C GLU A 333 5648 5599 5700 −8 55 47 C ATOM 783 O GLU A 333 6.661 29.064 −15.180 1.00 44.56 O ANISOU 783 O GLU A 333 5623 5595 5712 −41 123 71 O ATOM 784 N LYS A 334 6.230 27.892 −13.290 1.00 44.01 N ANISOU 784 N LYS A 334 5544 5539 5638 18 36 50 N ATOM 785 CA LYS A 334 6.873 26.659 −13.734 1.00 42.98 C ANISOU 785 CA LYS A 334 5422 5388 5518 15 17 31 C ATOM 786 CB LYS A 334 8.212 26.486 −13.006 1.00 43.19 C ANISOU 786 CB LYS A 334 5438 5429 5541 15 10 54 C ATOM 787 CG LYS A 334 9.342 27.415 −13.491 1.00 43.78 C ANISOU 787 CG LYS A 334 5486 5549 5597 −63 48 −57 C ATOM 788 CD LYS A 334 9.999 26.862 −14.756 1.00 47.27 C ANISOU 788 CD LYS A 334 6256 5959 5746 137 −131 −161 C ATOM 789 CE LYS A 334 11.210 27.670 −15.180 1.00 41.47 C ANISOU 789 CE LYS A 334 5573 4547 5633 −141 −13 547 C ATOM 790 NZ LYS A 334 10.822 28.944 −15.871 1.00 48.95 N ANISOU 790 NZ LYS A 334 5949 6476 6171 −108 −137 −288 N ATOM 791 C LYS A 334 5.942 25.479 −13.449 1.00 42.38 C ANISOU 791 C LYS A 334 5339 5347 5415 47 18 4 C ATOM 792 O LYS A 334 5.416 25.373 −12.342 1.00 41.36 O ANISOU 792 O LYS A 334 5235 5235 5242 88 46 19 O ATOM 793 N THR A 335 5.737 24.606 −14.448 1.00 42.28 N ANISOU 793 N THR A 335 5315 5358 5388 22 −3 5 N ATOM 794 CA THR A 335 4.887 23.413 −14.297 1.00 42.27 C ANISOU 794 CA THR A 335 5328 5336 5394 20 −27 −22 C ATOM 795 CB THR A 335 3.669 23.442 −15.297 1.00 42.66 C ANISOU 795 CB THR A 335 5389 5366 5451 24 −40 7 C ATOM 796 OG1 THR A 335 2.797 24.540 −14.973 1.00 43.84 O ANISOU 796 OG1 THR A 335 5548 5558 5551 61 −31 −90 O ATOM 797 CG2 THR A 335 2.849 22.137 −15.256 1.00 42.03 C ANISOU 797 CG2 THR A 335 5251 5336 5381 1 −34 33 C ATOM 798 C THR A 335 5.691 22.094 −14.399 1.00 42.02 C ANISOU 798 C THR A 335 5275 5343 5344 6 −2 3 C ATOM 799 O THR A 335 6.591 21.962 −15.233 1.00 42.24 O ANISOU 799 O THR A 335 5317 5422 5307 −6 27 −4 O ATOM 800 N ILE A 336 5.364 21.130 −13.536 1.00 41.53 N ANISOU 800 N ILE A 336 5220 5253 5307 −22 0 −2 N ATOM 801 CA ILE A 336 6.033 19.830 −13.521 1.00 41.14 C ANISOU 801 CA ILE A 336 5192 5158 5280 −5 −6 −31 C ATOM 802 CB ILE A 336 7.112 19.767 −12.399 1.00 41.49 C ANISOU 802 CB ILE A 336 5260 5196 5307 8 7 −66 C ATOM 803 CG1 ILE A 336 8.130 18.656 −12.661 1.00 41.08 C ANISOU 803 CG1 ILE A 336 5207 5068 5332 72 −8 −97 C ATOM 804 CD1 ILE A 336 9.561 19.137 −12.634 1.00 40.27 C ANISOU 804 CD1 ILE A 336 5179 4874 5247 −8 −10 −97 C ATOM 805 CG2 ILE A 336 6.480 19.580 −11.011 1.00 42.34 C ANISOU 805 CG2 ILE A 336 5379 5259 5448 −35 90 0 C ATOM 806 C ILE A 336 5.046 18.664 −13.378 1.00 40.65 C ANISOU 806 C ILE A 336 5139 5100 5203 −7 0 −31 C ATOM 807 O ILE A 336 4.003 18.801 −12.761 1.00 41.39 O ANISOU 807 O ILE A 336 5205 5207 5311 −38 −23 −17 O ATOM 808 N SER A 337 5.387 17.522 −13.967 1.00 39.94 N ANISOU 808 N SER A 337 5084 5004 5084 −68 −53 −17 N ATOM 809 CA SER A 337 4.678 16.250 −13.746 1.00 38.58 C ANISOU 809 CA SER A 337 4946 4829 4883 −42 −91 −14 C ATOM 810 CB SER A 337 3.495 16.104 −14.717 1.00 38.30 C ANISOU 810 CB SER A 337 4878 4805 4869 −19 −98 −9 C ATOM 811 OG SER A 337 3.996 15.979 −16.037 1.00 37.12 O ANISOU 811 OG SER A 337 4890 4556 4658 −36 −329 74 O ATOM 812 C SER A 337 5.671 15.117 −13.992 1.00 37.26 C ANISOU 812 C SER A 337 4761 4669 4727 −28 −55 −53 C ATOM 813 O SER A 337 6.763 15.356 −14.454 1.00 36.64 O ANISOU 813 O SER A 337 4712 4560 4646 −72 −77 −119 O ATOM 814 N LYS A 338 5.259 13.888 −13.696 1.00 36.86 N ANISOU 814 N LYS A 338 4689 4665 4651 34 −19 −58 N ATOM 815 CA LYS A 338 5.995 12.667 −14.048 1.00 36.00 C ANISOU 815 CA LYS A 338 4571 4534 4570 −3 −18 −52 C ATOM 816 CB LYS A 338 5.191 11.468 −13.542 1.00 35.29 C ANISOU 816 CB LYS A 338 4455 4494 4457 5 24 −42 C ATOM 817 CG LYS A 338 5.828 10.099 −13.681 1.00 33.67 C ANISOU 817 CG LYS A 338 4244 4375 4173 27 −21 32 C ATOM 818 CD LYS A 338 4.832 8.999 −13.385 1.00 30.62 C ANISOU 818 CD LYS A 338 3883 4010 3741 34 −66 1 C ATOM 819 CE LYS A 338 3.843 8.803 −14.522 1.00 29.42 C ANISOU 819 CE LYS A 338 3949 3572 3656 25 104 81 C ATOM 820 NZ LYS A 338 4.576 8.396 −15.745 1.00 27.21 N ANISOU 820 NZ LYS A 338 3843 3118 3374 −83 −117 132 N ATOM 821 C LYS A 338 6.258 12.571 −15.572 1.00 35.50 C ANISOU 821 C LYS A 338 4535 4440 4512 −21 −20 −80 C ATOM 822 O LYS A 338 5.479 13.073 −16.374 1.00 34.30 O ANISOU 822 O LYS A 338 4439 4209 4381 −34 4 −116 O ATOM 823 N ALA A 339 7.366 11.950 −15.968 1.00 35.70 N ANISOU 823 N ALA A 339 4573 4471 4517 −35 −30 −65 N ATOM 824 CA ALA A 339 7.638 11.750 −17.393 1.00 36.15 C ANISOU 824 CA ALA A 339 4615 4521 4599 −15 −13 −58 C ATOM 825 CB ALA A 339 8.837 10.811 −17.606 1.00 36.05 C ANISOU 825 CB ALA A 339 4546 4589 4559 −45 −58 −37 C ATOM 826 C ALA A 339 6.373 11.204 −18.084 1.00 36.01 C ANISOU 826 C ALA A 339 4591 4506 4583 −12 −7 −66 C ATOM 827 O ALA A 339 5.767 10.235 −17.626 1.00 35.29 O ANISOU 827 O ALA A 339 4495 4403 4508 20 0 −69 O ATOM 828 N LYS A 340 5.957 11.871 −19.150 1.00 36.06 N ANISOU 828 N LYS A 340 4636 4483 4580 −50 15 −31 N ATOM 829 CA LYS A 340 4.776 11.478 −19.901 1.00 36.79 C ANISOU 829 CA LYS A 340 4727 4622 4626 −22 −2 −23 C ATOM 830 CB LYS A 340 4.201 12.675 −20.674 1.00 37.11 C ANISOU 830 CB LYS A 340 4729 4625 4746 −33 22 −34 C ATOM 831 CG LYS A 340 3.417 13.646 −19.808 1.00 38.15 C ANISOU 831 CG LYS A 340 4951 4783 4759 −17 8 −23 C ATOM 832 CD LYS A 340 3.035 14.930 −20.552 1.00 38.31 C ANISOU 832 CD LYS A 340 4857 4845 4852 −53 −56 −11 C ATOM 833 CE LYS A 340 2.454 15.928 −19.583 1.00 39.66 C ANISOU 833 CE LYS A 340 4859 5039 5171 67 −10 −26 C ATOM 834 NZ LYS A 340 2.557 17.333 −20.080 1.00 42.09 N ANISOU 834 NZ LYS A 340 5215 5411 5363 −28 21 40 N ATOM 835 C LYS A 340 5.120 10.297 −20.833 1.00 36.19 C ANISOU 835 C LYS A 340 4615 4591 4545 0 0 −14 C ATOM 836 O LYS A 340 6.286 9.917 −20.940 1.00 36.01 O ANISOU 836 O LYS A 340 4613 4608 4461 −17 −8 −9 O ATOM 837 N GLY A 341 4.109 9.717 −21.486 1.00 35.13 N ANISOU 837 N GLY A 341 4487 4443 4417 −7 9 −27 N ATOM 838 CA GLY A 341 4.315 8.552 −22.351 1.00 33.53 C ANISOU 838 CA GLY A 341 4232 4341 4165 28 −19 20 C ATOM 839 C GLY A 341 3.679 7.281 −21.845 1.00 32.52 C ANISOU 839 C GLY A 341 4085 4155 4114 71 −32 −34 C ATOM 840 O GLY A 341 3.552 7.075 −20.628 1.00 31.86 O ANISOU 840 O GLY A 341 3925 4073 4106 82 −44 −75 O ATOM 841 N GLN A 342 3.276 6.427 −22.781 1.00 31.81 N ANISOU 841 N GLN A 342 4021 4126 3939 78 −15 −18 N ATOM 842 CA GLN A 342 2.697 5.123 −22.472 1.00 31.42 C ANISOU 842 CA GLN A 342 4041 3988 3909 45 −37 −59 C ATOM 843 CB GLN A 342 2.283 4.401 −23.770 1.00 31.69 C ANISOU 843 CB GLN A 342 4054 3993 3992 79 −26 −40 C ATOM 844 CG GLN A 342 1.017 4.931 −24.463 1.00 30.27 C ANISOU 844 CG GLN A 342 4081 3823 3597 2 −53 −130 C ATOM 845 CD GLN A 342 −0.282 4.551 −23.748 1.00 28.05 C ANISOU 845 CD GLN A 342 3804 3406 3445 149 −27 92 C ATOM 846 OE1 GLN A 342 −0.345 3.562 −22.997 1.00 31.57 O ANISOU 846 OE1 GLN A 342 4346 4036 3611 80 115 −150 O ATOM 847 NE2 GLN A 342 −1.324 5.329 −23.982 1.00 27.78 N ANISOU 847 NE2 GLN A 342 3742 3685 3128 63 86 28 N ATOM 848 C GLN A 342 3.699 4.278 −21.678 1.00 31.71 C ANISOU 848 C GLN A 342 4108 3972 3968 2 −33 −40 C ATOM 849 O GLN A 342 4.828 4.083 −22.128 1.00 31.42 O ANISOU 849 O GLN A 342 4162 3884 3891 −39 −112 −23 O ATOM 850 N PRO A 343 3.301 3.785 −20.489 1.00 32.23 N ANISOU 850 N PRO A 343 4160 4035 4051 10 −31 −23 N ATOM 851 CA PRO A 343 4.181 2.960 −19.638 1.00 32.69 C ANISOU 851 CA PRO A 343 4213 4109 4098 −3 −4 6 C ATOM 852 CB PRO A 343 3.380 2.837 −18.349 1.00 32.19 C ANISOU 852 CB PRO A 343 4159 4011 4060 30 −13 10 C ATOM 853 CG PRO A 343 1.964 2.866 −18.832 1.00 32.66 C ANISOU 853 CG PRO A 343 4242 4047 4116 0 21 −24 C ATOM 854 CD PRO A 343 1.968 3.940 −19.879 1.00 32.17 C ANISOU 854 CD PRO A 343 4181 4052 3987 16 −21 −13 C ATOM 855 C PRO A 343 4.431 1.555 −20.220 1.00 33.20 C ANISOU 855 C PRO A 343 4284 4187 4141 −16 −2 31 C ATOM 856 O PRO A 343 3.523 0.944 −20.760 1.00 32.66 O ANISOU 856 O PRO A 343 4246 4100 4061 −46 −31 42 O ATOM 857 N ARG A 344 5.661 1.059 −20.113 1.00 34.11 N ANISOU 857 N ARG A 344 4385 4381 4194 −20 −12 7 N ATOM 858 CA ARG A 344 5.966 −0.318 −20.495 1.00 34.88 C ANISOU 858 CA ARG A 344 4443 4484 4326 4 −8 −16 C ATOM 859 CB ARG A 344 6.837 −0.389 −21.766 1.00 35.47 C ANISOU 859 CB ARG A 344 4420 4544 4511 −12 −30 −52 C ATOM 860 CG ARG A 344 6.369 0.413 −22.984 1.00 35.46 C ANISOU 860 CG ARG A 344 4558 4556 4357 −10 103 −41 C ATOM 861 CD ARG A 344 7.187 −0.019 −24.224 1.00 42.30 C ANISOU 861 CD ARG A 344 5121 5594 5357 27 −186 −291 C ATOM 862 NE ARG A 344 7.453 1.068 −25.179 1.00 34.97 N ANISOU 862 NE ARG A 344 4759 4835 3690 −15 779 214 N ATOM 863 CZ ARG A 344 8.223 0.943 −26.268 1.00 46.83 C ANISOU 863 CZ ARG A 344 5377 6384 6029 −370 −579 −303 C ATOM 864 NH1 ARG A 344 8.798 −0.225 −26.541 1.00 36.80 N ANISOU 864 NH1 ARG A 344 4533 4393 5057 477 227 −349 N ATOM 865 NH2 ARG A 344 8.406 1.976 −27.085 1.00 32.95 N ANISOU 865 NH2 ARG A 344 4473 3988 4058 −185 138 349 N ATOM 866 C ARG A 344 6.652 −1.014 −19.326 1.00 35.55 C ANISOU 866 C ARG A 344 4503 4565 4439 −1 2 −55 C ATOM 867 O ARG A 344 7.600 −0.490 −18.751 1.00 35.51 O ANISOU 867 O ARG A 344 4476 4590 4426 24 −11 −37 O ATOM 868 N GLU A 345 6.144 −2.186 −18.971 1.00 35.97 N ANISOU 868 N GLU A 345 4588 4586 4489 −8 −41 −78 N ATOM 869 CA GLU A 345 6.656 −2.995 −17.880 1.00 36.16 C ANISOU 869 CA GLU A 345 4630 4626 4481 3 −56 −31 C ATOM 870 CB GLU A 345 5.668 −4.114 −17.573 1.00 36.66 C ANISOU 870 CB GLU A 345 4710 4666 4553 2 −39 −45 C ATOM 871 CG GLU A 345 5.939 −4.802 −16.249 1.00 38.92 C ANISOU 871 CG GLU A 345 5030 4961 4795 50 −70 23 C ATOM 872 CD GLU A 345 5.466 −6.244 −16.197 1.00 39.25 C ANISOU 872 CD GLU A 345 4949 4976 4987 −33 −88 12 C ATOM 873 OE1 GLU A 345 6.215 −7.086 −15.680 1.00 41.62 O ANISOU 873 OE1 GLU A 345 5509 5056 5247 −30 46 −48 O ATOM 874 OE2 GLU A 345 4.360 −6.544 −16.660 1.00 41.29 O ANISOU 874 OE2 GLU A 345 5302 5254 5132 24 −6 −54 O ATOM 875 C GLU A 345 8.014 −3.607 −18.213 1.00 36.11 C ANISOU 875 C GLU A 345 4648 4610 4459 −14 −51 −17 C ATOM 876 O GLU A 345 8.151 −4.270 −19.242 1.00 35.55 O ANISOU 876 O GLU A 345 4678 4510 4316 −23 −92 −1 O ATOM 877 N PRO A 346 9.029 −3.396 −17.338 1.00 35.96 N ANISOU 877 N PRO A 346 4575 4638 4446 −16 −46 −18 N ATOM 878 CA PRO A 346 10.320 −4.041 −17.561 1.00 35.46 C ANISOU 878 CA PRO A 346 4492 4581 4399 4 −42 −18 C ATOM 879 CB PRO A 346 11.182 −3.552 −16.380 1.00 35.73 C ANISOU 879 CB PRO A 346 4524 4597 4452 12 −21 4 C ATOM 880 CG PRO A 346 10.228 −3.061 −15.358 1.00 35.62 C ANISOU 880 CG PRO A 346 4572 4580 4380 38 −12 −22 C ATOM 881 CD PRO A 346 9.046 −2.540 −16.136 1.00 35.89 C ANISOU 881 CD PRO A 346 4545 4611 4479 −7 −43 −41 C ATOM 882 C PRO A 346 10.299 −5.561 −17.570 1.00 35.18 C ANISOU 882 C PRO A 346 4416 4581 4370 −24 −79 −24 C ATOM 883 O PRO A 346 9.494 −6.194 −16.877 1.00 34.62 O ANISOU 883 O PRO A 346 4353 4580 4219 −12 −186 −66 O ATOM 884 N GLN A 347 11.219 −6.120 −18.349 1.00 35.18 N ANISOU 884 N GLN A 347 4543 4504 4317 −46 −63 −72 N ATOM 885 CA GLN A 347 11.631 −7.507 −18.254 1.00 35.53 C ANISOU 885 CA GLN A 347 4553 4530 4414 0 −45 −78 C ATOM 886 CB GLN A 347 11.789 −8.075 −19.670 1.00 36.10 C ANISOU 886 CB GLN A 347 4629 4569 4515 5 −25 −114 C ATOM 887 CG GLN A 347 10.539 −7.907 −20.521 1.00 37.59 C ANISOU 887 CG GLN A 347 4887 4687 4707 85 −90 −113 C ATOM 888 CD GLN A 347 10.854 −7.408 −21.902 1.00 40.93 C ANISOU 888 CD GLN A 347 5327 5075 5147 −59 −3 9 C ATOM 889 OE1 GLN A 347 11.724 −7.946 −22.587 1.00 42.38 O ANISOU 889 OE1 GLN A 347 5289 5375 5436 61 68 117 O ATOM 890 NE2 GLN A 347 10.135 −6.381 −22.337 1.00 41.52 N ANISOU 890 NE2 GLN A 347 5299 5090 5385 92 −137 −75 N ATOM 891 C GLN A 347 12.969 −7.538 −17.513 1.00 34.81 C ANISOU 891 C GLN A 347 4451 4436 4337 −31 7 −68 C ATOM 892 O GLN A 347 13.907 −6.895 −17.937 1.00 34.34 O ANISOU 892 O GLN A 347 4438 4322 4286 −90 −26 −42 O ATOM 893 N VAL A 348 13.034 −8.283 −16.409 1.00 34.50 N ANISOU 893 N VAL A 348 4388 4352 4366 −33 16 −8 N ATOM 894 CA VAL A 348 14.195 −8.336 −15.520 1.00 34.25 C ANISOU 894 CA VAL A 348 4345 4345 4324 −31 17 −6 C ATOM 895 CB VAL A 348 13.798 −8.061 −14.027 1.00 34.35 C ANISOU 895 CB VAL A 348 4334 4344 4372 −35 28 27 C ATOM 896 CG1 VAL A 348 14.988 −8.250 −13.096 1.00 33.51 C ANISOU 896 CG1 VAL A 348 4238 4355 4138 −36 50 −12 C ATOM 897 CG2 VAL A 348 13.222 −6.668 −13.844 1.00 33.93 C ANISOU 897 CG2 VAL A 348 4281 4316 4294 5 17 −7 C ATOM 898 C VAL A 348 14.833 −9.720 −15.601 1.00 34.64 C ANISOU 898 C VAL A 348 4404 4383 4373 −32 6 27 C ATOM 899 O VAL A 348 14.156 −10.730 −15.359 1.00 34.49 O ANISOU 899 O VAL A 348 4366 4418 4317 −11 −34 44 O ATOM 900 N TYR A 349 16.126 −9.769 −15.935 1.00 34.98 N ANISOU 900 N TYR A 349 4437 4393 4459 −54 12 32 N ATOM 901 CA TYR A 349 16.835 −11.040 −16.125 1.00 35.60 C ANISOU 901 CA TYR A 349 4504 4476 4547 −57 −29 71 C ATOM 902 CB TYR A 349 17.128 −11.334 −17.612 1.00 33.85 C ANISOU 902 CB TYR A 349 4148 4389 4323 −53 −206 −7 C ATOM 903 CG TYR A 349 15.930 −11.322 −18.532 1.00 37.15 C ANISOU 903 CG TYR A 349 4880 4508 4724 99 192 −36 C ATOM 904 CD1 TYR A 349 14.882 −12.237 −18.364 1.00 33.27 C ANISOU 904 CD1 TYR A 349 4290 4016 4335 −10 −59 97 C ATOM 905 CE1 TYR A 349 13.785 −12.242 −19.206 1.00 31.99 C ANISOU 905 CE1 TYR A 349 4268 3911 3974 66 −115 123 C ATOM 906 CZ TYR A 349 13.707 −11.324 −20.253 1.00 36.97 C ANISOU 906 CZ TYR A 349 5013 4524 4509 156 357 −207 C ATOM 907 OH TYR A 349 12.592 −11.340 −21.085 1.00 32.56 O ANISOU 907 OH TYR A 349 3951 4398 4022 −11 −218 −91 O ATOM 908 CE2 TYR A 349 14.726 −10.404 −20.457 1.00 31.78 C ANISOU 908 CE2 TYR A 349 4079 4051 3944 −130 10 −26 C ATOM 909 CD2 TYR A 349 15.850 −10.416 −19.605 1.00 34.01 C ANISOU 909 CD2 TYR A 349 4476 4272 4172 69 −61 92 C ATOM 910 C TYR A 349 18.143 −11.028 −15.368 1.00 36.74 C ANISOU 910 C TYR A 349 4676 4594 4687 −68 −58 59 C ATOM 911 O TYR A 349 18.887 −10.037 −15.418 1.00 37.20 O ANISOU 911 O TYR A 349 4797 4592 4743 −80 −156 107 O ATOM 912 N THR A 350 18.432 −12.130 −14.680 1.00 37.32 N ANISOU 912 N THR A 350 4742 4621 4817 −56 −66 36 N ATOM 913 CA THR A 350 19.718 −12.304 −14.026 1.00 38.14 C ANISOU 913 CA THR A 350 4825 4721 4943 −21 −81 20 C ATOM 914 CB THR A 350 19.559 −12.839 −12.597 1.00 38.40 C ANISOU 914 CB THR A 350 4860 4761 4966 −31 −61 7 C ATOM 915 OG1 THR A 350 18.731 −14.018 −12.598 1.00 39.46 O ANISOU 915 OG1 THR A 350 4927 4782 5282 −54 −167 −97 O ATOM 916 CG2 THR A 350 18.928 −11.782 −11.709 1.00 37.43 C ANISOU 916 CG2 THR A 350 4800 4532 4887 17 −112 −76 C ATOM 917 C THR A 350 20.611 −13.217 −14.868 1.00 38.74 C ANISOU 917 C THR A 350 4927 4769 5022 1 −79 18 C ATOM 918 O THR A 350 20.123 −14.185 −15.444 1.00 38.86 O ANISOU 918 O THR A 350 4914 4739 5112 8 −126 13 O ATOM 919 N LEU A 351 21.898 −12.871 −14.979 1.00 39.30 N ANISOU 919 N LEU A 351 4977 4875 5078 7 −62 40 N ATOM 920 CA LEU A 351 22.867 −13.658 −15.755 1.00 40.29 C ANISOU 920 CA LEU A 351 5136 5014 5156 0 −38 5 C ATOM 921 CB LEU A 351 23.357 −12.912 −17.004 1.00 40.56 C ANISOU 921 CB LEU A 351 5169 5086 5156 −3 −7 −12 C ATOM 922 CG LEU A 351 22.508 −11.927 −17.836 1.00 41.22 C ANISOU 922 CG LEU A 351 5191 5222 5247 −1 −7 19 C ATOM 923 CD1 LEU A 351 23.198 −11.617 −19.177 1.00 42.94 C ANISOU 923 CD1 LEU A 351 5594 5366 5355 47 57 −43 C ATOM 924 CD2 LEU A 351 21.117 −12.418 −18.099 1.00 41.57 C ANISOU 924 CD2 LEU A 351 5226 5213 5354 23 −32 −39 C ATOM 925 C LEU A 351 24.076 −14.028 −14.888 1.00 40.94 C ANISOU 925 C LEU A 351 5192 5092 5268 4 −33 −14 C ATOM 926 O LEU A 351 24.582 −13.189 −14.116 1.00 41.33 O ANISOU 926 O LEU A 351 5233 5217 5252 6 −14 2 O ATOM 927 N PRO A 352 24.561 −15.277 −15.020 1.00 41.37 N ANISOU 927 N PRO A 352 5252 5155 5309 −9 −38 −17 N ATOM 928 CA PRO A 352 25.657 −15.706 −14.168 1.00 41.43 C ANISOU 928 CA PRO A 352 5288 5166 5287 4 −22 0 C ATOM 929 CB PRO A 352 25.512 −17.225 −14.185 1.00 41.27 C ANISOU 929 CB PRO A 352 5261 5141 5277 −45 −23 −16 C ATOM 930 CG PRO A 352 25.003 −17.514 −15.565 1.00 41.74 C ANISOU 930 CG PRO A 352 5297 5231 5329 −13 −41 16 C ATOM 931 CD PRO A 352 24.147 −16.336 −15.966 1.00 41.31 C ANISOU 931 CD PRO A 352 5282 5099 5315 6 7 −17 C ATOM 932 C PRO A 352 27.006 −15.272 −14.759 1.00 41.59 C ANISOU 932 C PRO A 352 5340 5165 5297 −16 0 11 C ATOM 933 O PRO A 352 27.064 −14.880 −15.928 1.00 42.08 O ANISOU 933 O PRO A 352 5477 5215 5295 16 16 −12 O ATOM 934 N PRO A 353 28.089 −15.328 −13.967 1.00 41.79 N ANISOU 934 N PRO A 353 5347 5197 5334 −44 8 11 N ATOM 935 CA PRO A 353 29.375 −14.972 −14.556 1.00 42.47 C ANISOU 935 CA PRO A 353 5405 5319 5412 −10 26 −14 C ATOM 936 CB PRO A 353 30.363 −15.451 −13.503 1.00 42.11 C ANISOU 936 CB PRO A 353 5366 5274 5359 6 19 1 C ATOM 937 CG PRO A 353 29.616 −15.312 −12.210 1.00 41.83 C ANISOU 937 CG PRO A 353 5318 5207 5368 −12 18 34 C ATOM 938 CD PRO A 353 28.212 −15.662 −12.533 1.00 41.62 C ANISOU 938 CD PRO A 353 5287 5204 5320 −37 9 −1 C ATOM 939 C PRO A 353 29.664 −15.642 −15.914 1.00 43.38 C ANISOU 939 C PRO A 353 5539 5438 5506 −59 7 0 C ATOM 940 O PRO A 353 29.099 −16.686 −16.238 1.00 43.79 O ANISOU 940 O PRO A 353 5568 5453 5616 −92 −13 7 O ATOM 941 N SER A 354 30.550 −15.029 −16.692 1.00 44.02 N ANISOU 941 N SER A 354 5647 5507 5570 −62 32 25 N ATOM 942 CA SER A 354 31.029 −15.600 −17.947 1.00 44.25 C ANISOU 942 CA SER A 354 5684 5544 5581 −36 2 16 C ATOM 943 CB SER A 354 31.801 −14.533 −18.728 1.00 43.63 C ANISOU 943 CB SER A 354 5643 5426 5508 −42 5 44 C ATOM 944 OG SER A 354 32.102 −14.957 −20.044 1.00 43.43 O ANISOU 944 OG SER A 354 5647 5312 5541 −41 −107 63 O ATOM 945 C SER A 354 31.932 −16.816 −17.684 1.00 44.69 C ANISOU 945 C SER A 354 5752 5587 5640 −28 −2 53 C ATOM 946 O SER A 354 32.576 −16.911 −16.628 1.00 45.23 O ANISOU 946 O SER A 354 5780 5703 5700 −78 −15 91 O ATOM 947 N ARG A 355 31.978 −17.740 −18.639 1.00 45.17 N ANISOU 947 N ARG A 355 5825 5641 5696 −10 22 54 N ATOM 948 CA ARG A 355 32.923 −18.860 −18.570 1.00 45.90 C ANISOU 948 CA ARG A 355 5863 5750 5824 12 −2 65 C ATOM 949 CB ARG A 355 32.871 −19.735 −19.832 1.00 46.10 C ANISOU 949 CB ARG A 355 5934 5737 5845 20 17 36 C ATOM 950 CG ARG A 355 33.824 −20.954 −19.799 1.00 47.55 C ANISOU 950 CG ARG A 355 6059 5947 6060 67 78 50 C ATOM 951 CD ARG A 355 33.145 −22.221 −19.262 1.00 49.46 C ANISOU 951 CD ARG A 355 6372 5960 6459 −34 23 14 C ATOM 952 NE ARG A 355 34.055 −23.376 −19.231 1.00 51.31 N ANISOU 952 NE ARG A 355 6419 6227 6849 47 33 24 N ATOM 953 CZ ARG A 355 33.662 −24.659 −19.211 1.00 49.87 C ANISOU 953 CZ ARG A 355 5961 6130 6856 36 60 63 C ATOM 954 NH1 ARG A 355 32.368 −24.974 −19.225 1.00 51.01 N ANISOU 954 NH1 ARG A 355 6530 6058 6792 −50 14 64 N ATOM 955 NH2 ARG A 355 34.566 −25.636 −19.183 1.00 51.06 N ANISOU 955 NH2 ARG A 355 6527 6353 6518 −114 122 111 N ATOM 956 C ARG A 355 34.337 −18.338 −18.363 1.00 46.16 C ANISOU 956 C ARG A 355 5873 5790 5874 3 −27 85 C ATOM 957 O ARG A 355 35.002 −18.735 −17.402 1.00 46.73 O ANISOU 957 O ARG A 355 5996 5863 5893 12 −54 137 O ATOM 958 N GLU A 356 34.784 −17.436 −19.244 1.00 46.08 N ANISOU 958 N GLU A 356 5863 5771 5872 22 −47 101 N ATOM 959 CA GLU A 356 36.160 −16.914 −19.201 1.00 45.95 C ANISOU 959 CA GLU A 356 5824 5716 5915 −12 −25 83 C ATOM 960 CB GLU A 356 36.460 −16.024 −20.406 1.00 46.56 C ANISOU 960 CB GLU A 356 5923 5802 5963 −14 −29 55 C ATOM 961 CG GLU A 356 35.478 −16.076 −21.574 1.00 48.71 C ANISOU 961 CG GLU A 356 6157 6172 6178 −18 −52 −43 C ATOM 962 CD GLU A 356 35.388 −14.734 −22.325 1.00 44.20 C ANISOU 962 CD GLU A 356 5022 5601 6171 −315 −566 188 C ATOM 963 OE1 GLU A 356 36.355 −13.927 −22.227 1.00 50.80 O ANISOU 963 OE1 GLU A 356 6751 6418 6131 327 68 47 O ATOM 964 OE2 GLU A 356 34.348 −14.493 −23.006 1.00 52.20 O ANISOU 964 OE2 GLU A 356 7079 5919 6833 −107 191 −53 O ATOM 965 C GLU A 356 36.487 −16.133 −17.917 1.00 45.96 C ANISOU 965 C GLU A 356 5812 5714 5934 −1 −14 67 C ATOM 966 O GLU A 356 37.654 −16.022 −17.533 1.00 46.16 O ANISOU 966 O GLU A 356 5833 5670 6036 10 −15 75 O ATOM 967 N GLU A 357 35.465 −15.585 −17.259 1.00 46.00 N ANISOU 967 N GLU A 357 5832 5718 5928 −5 −1 46 N ATOM 968 CA GLU A 357 35.658 −14.822 −16.022 1.00 46.17 C ANISOU 968 CA GLU A 357 5858 5789 5893 −19 −29 37 C ATOM 969 CB GLU A 357 34.500 −13.832 −15.807 1.00 46.20 C ANISOU 969 CB GLU A 357 5855 5775 5922 −22 −31 25 C ATOM 970 CG GLU A 357 34.741 −12.830 −14.672 1.00 46.03 C ANISOU 970 CG GLU A 357 5890 5813 5786 −3 20 5 C ATOM 971 CD GLU A 357 33.511 −12.050 −14.288 1.00 45.84 C ANISOU 971 CD GLU A 357 5810 5736 5870 18 −15 23 C ATOM 972 OE1 GLU A 357 32.394 −12.574 −14.466 1.00 47.43 O ANISOU 972 OE1 GLU A 357 5997 5800 6223 −69 19 64 O ATOM 973 OE2 GLU A 357 33.665 −10.908 −13.793 1.00 47.18 O ANISOU 973 OE2 GLU A 357 6018 5915 5990 −47 69 47 O ATOM 974 C GLU A 357 35.791 −15.713 −14.789 1.00 46.48 C ANISOU 974 C GLU A 357 5902 5797 5962 2 −60 36 C ATOM 975 O GLU A 357 36.116 −15.242 −13.695 1.00 46.82 O ANISOU 975 O GLU A 357 5954 5880 5956 12 −81 46 O ATOM 976 N MET A 358 35.527 −17.000 −14.966 1.00 47.05 N ANISOU 976 N MET A 358 5945 5879 6053 −19 −52 49 N ATOM 977 CA MET A 358 35.559 −17.963 −13.864 1.00 47.74 C ANISOU 977 CA MET A 358 6017 5957 6164 −17 −69 67 C ATOM 978 CB MET A 358 34.728 −19.192 −14.238 1.00 48.21 C ANISOU 978 CB MET A 358 6105 5944 6269 −34 −75 93 C ATOM 979 CG MET A 358 33.208 −18.945 −14.199 1.00 49.31 C ANISOU 979 CG MET A 358 6165 6106 6465 −8 −14 71 C ATOM 980 SD MET A 358 32.552 −19.075 −12.527 1.00 52.44 S ANISOU 980 SD MET A 358 6730 6476 6717 −163 20 106 S ATOM 981 CE MET A 358 32.822 −17.454 −11.834 1.00 51.39 C ANISOU 981 CE MET A 358 6475 6527 6523 47 −35 165 C ATOM 982 C MET A 358 36.981 −18.353 −13.440 1.00 47.84 C ANISOU 982 C MET A 358 6037 5968 6170 −13 −76 65 C ATOM 983 O MET A 358 37.261 −19.524 −13.131 1.00 48.04 O ANISOU 983 O MET A 358 6128 5948 6176 −23 −92 42 O ATOM 984 N THR A 359 37.855 −17.348 −13.399 1.00 47.68 N ANISOU 984 N THR A 359 6033 5948 6134 −20 −77 59 N ATOM 985 CA THR A 359 39.290 −17.516 −13.163 1.00 47.64 C ANISOU 985 CA THR A 359 6020 5995 6085 0 −63 39 C ATOM 986 CB THR A 359 40.100 −17.070 −14.403 1.00 47.73 C ANISOU 986 CB THR A 359 6003 6022 6108 15 −50 37 C ATOM 987 OG1 THR A 359 39.769 −17.914 −15.522 1.00 49.09 O ANISOU 987 OG1 THR A 359 6271 6170 6209 91 −41 16 O ATOM 988 CG2 THR A 359 41.608 −17.130 −14.143 1.00 48.38 C ANISOU 988 CG2 THR A 359 6138 6071 6172 −8 −78 7 C ATOM 989 C THR A 359 39.722 −16.691 −11.958 1.00 47.42 C ANISOU 989 C THR A 359 5966 5981 6070 −5 −71 67 C ATOM 990 O THR A 359 40.618 −17.090 −11.219 1.00 47.99 O ANISOU 990 O THR A 359 6050 6064 6120 6 −66 108 O ATOM 991 N LYS A 360 39.081 −15.545 −11.763 1.00 46.86 N ANISOU 991 N LYS A 360 5868 5926 6009 11 −50 41 N ATOM 992 CA LYS A 360 39.494 −14.595 −10.743 1.00 47.00 C ANISOU 992 CA LYS A 360 5889 5971 5996 29 −30 61 C ATOM 993 CB LYS A 360 39.110 −13.153 −11.142 1.00 46.95 C ANISOU 993 CB LYS A 360 5886 5968 5983 21 −5 46 C ATOM 994 CG LYS A 360 38.907 −12.927 −12.637 1.00 47.63 C ANISOU 994 CG LYS A 360 5963 6082 6050 61 −16 111 C ATOM 995 CD LYS A 360 40.223 −12.855 −13.426 1.00 50.53 C ANISOU 995 CD LYS A 360 6261 6404 6531 60 21 9 C ATOM 996 CE LYS A 360 39.961 −12.875 −14.932 1.00 49.77 C ANISOU 996 CE LYS A 360 6180 6480 6250 30 132 33 C ATOM 997 NZ LYS A 360 41.160 −12.482 −15.751 1.00 53.54 N ANISOU 997 NZ LYS A 360 6756 6647 6937 76 9 −32 N ATOM 998 C LYS A 360 38.846 −15.001 −9.427 1.00 47.06 C ANISOU 998 C LYS A 360 5905 5954 6021 12 −44 84 C ATOM 999 O LYS A 360 38.141 −16.004 −9.374 1.00 47.87 O ANISOU 999 O LYS A 360 6011 6041 6135 7 −64 106 O ATOM 1000 N ASN A 361 39.081 −14.239 −8.368 1.00 46.80 N ANISOU 1000 N ASN A 361 5895 5940 5944 39 −18 64 N ATOM 1001 CA ASN A 361 38.505 −14.578 −7.076 1.00 47.13 C ANISOU 1001 CA ASN A 361 5932 5982 5992 48 −3 96 C ATOM 1002 CB ASN A 361 39.554 −14.470 −5.963 1.00 47.64 C ANISOU 1002 CB ASN A 361 5962 6070 6066 49 −24 69 C ATOM 1003 CG ASN A 361 40.414 −13.225 −6.085 1.00 48.35 C ANISOU 1003 CG ASN A 361 6032 6093 6242 20 57 53 C ATOM 1004 OD1 ASN A 361 39.932 −12.094 −5.891 1.00 50.52 O ANISOU 1004 OD1 ASN A 361 6442 6402 6351 180 54 178 O ATOM 1005 ND2 ASN A 361 41.709 −13.426 −6.389 1.00 49.41 N ANISOU 1005 ND2 ASN A 361 6268 6360 6143 31 14 44 N ATOM 1006 C ASN A 361 37.271 −13.744 −6.746 1.00 46.75 C ANISOU 1006 C ASN A 361 5840 5963 5960 64 −21 151 C ATOM 1007 O ASN A 361 36.510 −14.058 −5.815 1.00 47.14 O ANISOU 1007 O ASN A 361 5925 6049 5938 101 −35 168 O ATOM 1008 N GLN A 362 37.081 −12.666 −7.503 1.00 46.36 N ANISOU 1008 N GLN A 362 5775 5900 5936 28 11 116 N ATOM 1009 CA GLN A 362 35.802 −11.967 −7.500 1.00 45.32 C ANISOU 1009 CA GLN A 362 5680 5745 5794 29 −4 84 C ATOM 1010 CB GLN A 362 35.974 −10.507 −7.118 1.00 45.41 C ANISOU 1010 CB GLN A 362 5707 5735 5811 14 −8 83 C ATOM 1011 CG GLN A 362 36.150 −10.334 −5.621 1.00 45.58 C ANISOU 1011 CG GLN A 362 5724 5792 5802 −53 11 71 C ATOM 1012 CD GLN A 362 36.166 −8.891 −5.204 1.00 45.91 C ANISOU 1012 CD GLN A 362 5751 5817 5875 17 163 20 C ATOM 1013 OE1 GLN A 362 35.381 −8.478 −4.363 1.00 47.89 O ANISOU 1013 OE1 GLN A 362 6152 5957 6085 −12 83 107 O ATOM 1014 NE2 GLN A 362 37.063 −8.105 −5.797 1.00 48.21 N ANISOU 1014 NE2 GLN A 362 6100 5959 6259 38 0 10 N ATOM 1015 C GLN A 362 35.119 −12.139 −8.842 1.00 44.69 C ANISOU 1015 C GLN A 362 5577 5712 5689 45 2 97 C ATOM 1016 O GLN A 362 35.779 −12.142 −9.875 1.00 45.00 O ANISOU 1016 O GLN A 362 5562 5769 5766 68 40 92 O ATOM 1017 N VAL A 363 33.799 −12.329 −8.807 1.00 44.15 N ANISOU 1017 N VAL A 363 5530 5638 5605 38 −12 83 N ATOM 1018 CA VAL A 363 32.983 −12.486 −10.023 1.00 43.03 C ANISOU 1018 CA VAL A 363 5413 5445 5488 52 −6 43 C ATOM 1019 CB VAL A 363 32.402 −13.912 −10.140 1.00 43.18 C ANISOU 1019 CB VAL A 363 5453 5459 5492 67 13 28 C ATOM 1020 CG1 VAL A 363 33.520 −14.906 −10.390 1.00 43.95 C ANISOU 1020 CG1 VAL A 363 5470 5624 5603 82 29 48 C ATOM 1021 CG2 VAL A 363 31.600 −14.302 −8.882 1.00 42.30 C ANISOU 1021 CG2 VAL A 363 5359 5266 5446 80 −4 36 C ATOM 1022 C VAL A 363 31.853 −11.440 −10.120 1.00 42.52 C ANISOU 1022 C VAL A 363 5376 5398 5380 63 −27 59 C ATOM 1023 O VAL A 363 31.447 −10.834 −9.104 1.00 42.81 O ANISOU 1023 O VAL A 363 5403 5365 5495 71 −45 3 O ATOM 1024 N SER A 364 31.354 −11.252 −11.343 1.00 41.31 N ANISOU 1024 N SER A 364 5256 5207 5231 62 −5 118 N ATOM 1025 CA SER A 364 30.298 −10.280 −11.635 1.00 40.20 C ANISOU 1025 CA SER A 364 5150 5057 5065 36 −19 138 C ATOM 1026 CB SER A 364 30.652 −9.403 −12.850 1.00 39.70 C ANISOU 1026 CB SER A 364 5109 4974 5002 54 −65 171 C ATOM 1027 OG SER A 364 31.895 −8.732 −12.692 1.00 37.97 O ANISOU 1027 OG SER A 364 5007 4675 4744 119 −32 315 O ATOM 1028 C SER A 364 28.962 −10.967 −11.868 1.00 39.95 C ANISOU 1028 C SER A 364 5127 5016 5034 45 −31 160 C ATOM 1029 O SER A 364 28.831 −11.854 −12.735 1.00 39.44 O ANISOU 1029 O SER A 364 5142 4888 4955 54 −17 221 O ATOM 1030 N LEU A 365 27.981 −10.554 −11.071 1.00 39.87 N ANISOU 1030 N LEU A 365 5068 5037 5042 61 −21 140 N ATOM 1031 CA LEU A 365 26.589 −10.955 −11.269 1.00 39.99 C ANISOU 1031 CA LEU A 365 5074 5073 5045 9 −33 86 C ATOM 1032 CB LEU A 365 25.924 −11.321 −9.937 1.00 40.04 C ANISOU 1032 CB LEU A 365 5074 5098 5040 −11 −44 81 C ATOM 1033 CG LEU A 365 26.530 −12.451 −9.066 1.00 40.30 C ANISOU 1033 CG LEU A 365 5182 5030 5100 14 −46 30 C ATOM 1034 CD1 LEU A 365 25.665 −12.646 −7.874 1.00 39.40 C ANISOU 1034 CD1 LEU A 365 5149 4949 4869 −42 31 113 C ATOM 1035 CD2 LEU A 365 26.697 −13.782 −9.784 1.00 39.06 C ANISOU 1035 CD2 LEU A 365 5102 4928 4808 −9 −43 35 C ATOM 1036 C LEU A 365 25.801 −9.848 −11.985 1.00 39.79 C ANISOU 1036 C LEU A 365 4994 5066 5057 24 −48 75 C ATOM 1037 O LEU A 365 25.823 −8.677 −11.582 1.00 38.88 O ANISOU 1037 O LEU A 365 4910 4982 4882 74 −17 36 O ATOM 1038 N THR A 366 25.099 −10.244 −13.044 1.00 39.92 N ANISOU 1038 N THR A 366 5000 5095 5069 45 −33 66 N ATOM 1039 CA THR A 366 24.421 −9.292 −13.933 1.00 39.88 C ANISOU 1039 CA THR A 366 4988 5034 5128 11 −42 67 C ATOM 1040 CB THR A 366 24.903 −9.487 −15.393 1.00 39.60 C ANISOU 1040 CB THR A 366 4964 5029 5051 11 −16 62 C ATOM 1041 OG1 THR A 366 26.320 −9.308 −15.459 1.00 38.90 O ANISOU 1041 OG1 THR A 366 5015 4949 4816 −20 −181 162 O ATOM 1042 CG2 THR A 366 24.235 −8.512 −16.335 1.00 38.62 C ANISOU 1042 CG2 THR A 366 4770 4839 5062 −13 46 66 C ATOM 1043 C THR A 366 22.871 −9.311 −13.869 1.00 39.94 C ANISOU 1043 C THR A 366 4957 5035 5180 37 −21 65 C ATOM 1044 O THR A 366 22.232 −10.335 −14.103 1.00 39.93 O ANISOU 1044 O THR A 366 4869 5040 5262 43 12 60 O ATOM 1045 N CYS A 367 22.280 −8.161 −13.565 1.00 39.76 N ANISOU 1045 N CYS A 367 4930 5062 5115 29 −27 86 N ATOM 1046 CA CYS A 367 20.852 −7.960 −13.805 1.00 39.32 C ANISOU 1046 CA CYS A 367 4931 4985 5022 26 −8 80 C ATOM 1047 CB CYS A 367 20.233 −7.205 −12.634 1.00 39.39 C ANISOU 1047 CB CYS A 367 4944 5076 4946 21 −2 117 C ATOM 1048 SG CYS A 367 18.472 −7.483 −12.391 1.00 39.53 S ANISOU 1048 SG CYS A 367 4830 5094 5094 24 −129 170 S ATOM 1049 C CYS A 367 20.601 −7.210 −15.139 1.00 38.81 C ANISOU 1049 C CYS A 367 4865 4957 4923 15 −1 46 C ATOM 1050 O CYS A 367 21.014 −6.067 −15.311 1.00 38.63 O ANISOU 1050 O CYS A 367 4817 5047 4814 9 43 133 O ATOM 1051 N LEU A 368 19.929 −7.861 −16.076 1.00 38.38 N ANISOU 1051 N LEU A 368 4830 4891 4859 14 7 46 N ATOM 1052 CA LEU A 368 19.456 −7.190 −17.282 1.00 37.80 C ANISOU 1052 CA LEU A 368 4781 4779 4800 −21 24 8 C ATOM 1053 CB LEU A 368 19.625 −8.079 −18.516 1.00 37.58 C ANISOU 1053 CB LEU A 368 4733 4775 4770 −33 5 0 C ATOM 1054 CG LEU A 368 18.982 −7.599 −19.827 1.00 37.13 C ANISOU 1054 CG LEU A 368 4703 4665 4738 −43 63 −35 C ATOM 1055 CD1 LEU A 368 19.398 −6.178 −20.237 1.00 34.47 C ANISOU 1055 CD1 LEU A 368 4419 4312 4366 −26 110 −219 C ATOM 1056 CD2 LEU A 368 19.275 −8.606 −20.956 1.00 37.22 C ANISOU 1056 CD2 LEU A 368 4639 4690 4810 −62 −1 −69 C ATOM 1057 C LEU A 368 17.999 −6.740 −17.158 1.00 37.59 C ANISOU 1057 C LEU A 368 4799 4734 4747 −15 12 21 C ATOM 1058 O LEU A 368 17.086 −7.556 −17.070 1.00 38.09 O ANISOU 1058 O LEU A 368 4826 4762 4883 0 0 19 O ATOM 1059 N VAL A 369 17.785 −5.433 −17.182 1.00 36.66 N ANISOU 1059 N VAL A 369 4710 4628 4590 −14 8 −31 N ATOM 1060 CA VAL A 369 16.437 −4.899 −17.150 1.00 35.51 C ANISOU 1060 CA VAL A 369 4618 4432 4441 −32 24 −25 C ATOM 1061 CB VAL A 369 16.240 −3.901 −15.978 1.00 35.04 C ANISOU 1061 CB VAL A 369 4579 4397 4336 −16 9 −42 C ATOM 1062 CG1 VAL A 369 14.774 −3.571 −15.809 1.00 32.99 C ANISOU 1062 CG1 VAL A 369 4372 4088 4074 −154 64 −67 C ATOM 1063 CG2 VAL A 369 16.828 −4.445 −14.690 1.00 33.45 C ANISOU 1063 CG2 VAL A 369 4399 4030 4280 −68 82 −84 C ATOM 1064 C VAL A 369 16.149 −4.214 −18.495 1.00 35.59 C ANISOU 1064 C VAL A 369 4633 4477 4411 −5 55 −52 C ATOM 1065 O VAL A 369 16.819 −3.261 −18.862 1.00 33.94 O ANISOU 1065 O VAL A 369 4433 4347 4113 −86 75 14 O ATOM 1066 N LYS A 370 15.142 −4.699 −19.212 1.00 35.68 N ANISOU 1066 N LYS A 370 4629 4439 4486 5 61 −52 N ATOM 1067 CA LYS A 370 14.862 −4.155 −20.524 1.00 36.56 C ANISOU 1067 CA LYS A 370 4733 4538 4619 −4 54 −72 C ATOM 1068 CB LYS A 370 15.582 −4.963 −21.616 1.00 37.40 C ANISOU 1068 CB LYS A 370 4812 4631 4766 21 15 −67 C ATOM 1069 CG LYS A 370 14.926 −6.255 −22.047 1.00 38.42 C ANISOU 1069 CG LYS A 370 4880 4762 4953 −43 −18 −43 C ATOM 1070 CD LYS A 370 15.576 −6.722 −23.346 1.00 38.00 C ANISOU 1070 CD LYS A 370 4710 5016 4710 −111 −78 −144 C ATOM 1071 CE LYS A 370 14.530 −7.210 −24.325 1.00 42.54 C ANISOU 1071 CE LYS A 370 5280 5298 5583 88 107 −138 C ATOM 1072 NZ LYS A 370 14.737 −6.578 −25.665 1.00 40.11 N ANISOU 1072 NZ LYS A 370 5090 5259 4890 −7 15 108 N ATOM 1073 C LYS A 370 13.393 −3.913 −20.847 1.00 36.36 C ANISOU 1073 C LYS A 370 4750 4479 4586 26 55 −79 C ATOM 1074 O LYS A 370 12.496 −4.426 −20.174 1.00 35.86 O ANISOU 1074 O LYS A 370 4750 4336 4537 38 36 −46 O ATOM 1075 N GLY A 371 13.164 −3.101 −21.873 1.00 36.40 N ANISOU 1075 N GLY A 371 4754 4514 4560 14 101 −70 N ATOM 1076 CA GLY A 371 11.821 −2.792 −22.348 1.00 36.43 C ANISOU 1076 CA GLY A 371 4719 4559 4561 16 44 −51 C ATOM 1077 C GLY A 371 10.981 −1.871 −21.467 1.00 36.58 C ANISOU 1077 C GLY A 371 4751 4566 4581 22 1 −59 C ATOM 1078 O GLY A 371 9.739 −1.850 −21.601 1.00 36.50 O ANISOU 1078 O GLY A 371 4704 4546 4617 42 12 −66 O ATOM 1079 N PHE A 372 11.624 −1.118 −20.566 1.00 35.97 N ANISOU 1079 N PHE A 372 4631 4483 4552 −15 −16 −47 N ATOM 1080 CA PHE A 372 10.853 −0.250 −19.635 1.00 34.98 C ANISOU 1080 CA PHE A 372 4543 4431 4315 −24 −65 −38 C ATOM 1081 CB PHE A 372 11.347 −0.338 −18.188 1.00 34.15 C ANISOU 1081 CB PHE A 372 4462 4305 4208 −30 −25 7 C ATOM 1082 CG PHE A 372 12.732 0.220 −17.952 1.00 31.81 C ANISOU 1082 CG PHE A 372 4269 4040 3776 7 34 78 C ATOM 1083 CD1 PHE A 372 12.902 1.534 −17.521 1.00 29.99 C ANISOU 1083 CD1 PHE A 372 4046 4049 3299 −75 66 87 C ATOM 1084 CE1 PHE A 372 14.158 2.046 −17.270 1.00 29.52 C ANISOU 1084 CE1 PHE A 372 4091 3871 3254 −37 73 41 C ATOM 1085 CZ PHE A 372 15.278 1.245 −17.432 1.00 30.63 C ANISOU 1085 CZ PHE A 372 4204 3924 3509 −6 99 84 C ATOM 1086 CE2 PHE A 372 15.126 −0.073 −17.842 1.00 30.13 C ANISOU 1086 CE2 PHE A 372 4163 4003 3281 −10 108 120 C ATOM 1087 CD2 PHE A 372 13.849 −0.580 −18.094 1.00 29.81 C ANISOU 1087 CD2 PHE A 372 4076 3919 3330 −84 142 106 C ATOM 1088 C PHE A 372 10.664 1.198 −20.081 1.00 34.59 C ANISOU 1088 C PHE A 372 4487 4398 4257 −43 −112 −70 C ATOM 1089 O PHE A 372 11.487 1.745 −20.787 1.00 33.11 O ANISOU 1089 O PHE A 372 4403 4216 3961 −15 −136 −82 O ATOM 1090 N TYR A 373 9.549 1.783 −19.647 1.00 34.91 N ANISOU 1090 N TYR A 373 4521 4486 4255 −49 −176 −43 N ATOM 1091 CA TYR A 373 9.193 3.175 −19.910 1.00 35.31 C ANISOU 1091 CA TYR A 373 4589 4543 4284 −16 −143 −35 C ATOM 1092 CB TYR A 373 8.567 3.358 −21.307 1.00 35.68 C ANISOU 1092 CB TYR A 373 4580 4691 4283 −10 −157 −87 C ATOM 1093 CG TYR A 373 8.621 4.789 −21.762 1.00 34.18 C ANISOU 1093 CG TYR A 373 4411 4534 4041 −21 −162 −10 C ATOM 1094 CD1 TYR A 373 9.690 5.243 −22.509 1.00 34.45 C ANISOU 1094 CD1 TYR A 373 4413 4527 4147 23 −212 −54 C ATOM 1095 CE1 TYR A 373 9.774 6.554 −22.912 1.00 34.42 C ANISOU 1095 CE1 TYR A 373 4358 4572 4148 −101 −173 −16 C ATOM 1096 CZ TYR A 373 8.780 7.423 −22.558 1.00 34.48 C ANISOU 1096 CZ TYR A 373 4422 4435 4243 −45 −91 0 C ATOM 1097 OH TYR A 373 8.873 8.715 −22.969 1.00 36.84 O ANISOU 1097 OH TYR A 373 4769 4819 4407 −28 −199 64 O ATOM 1098 CE2 TYR A 373 7.702 7.007 −21.797 1.00 35.52 C ANISOU 1098 CE2 TYR A 373 4524 4712 4258 83 −192 −76 C ATOM 1099 CD2 TYR A 373 7.622 5.698 −21.414 1.00 33.27 C ANISOU 1099 CD2 TYR A 373 4467 4373 3798 −92 −214 63 C ATOM 1100 C TYR A 373 8.202 3.675 −18.866 1.00 35.10 C ANISOU 1100 C TYR A 373 4544 4556 4234 −5 −101 20 C ATOM 1101 O TYR A 373 7.290 2.959 −18.526 1.00 35.49 O ANISOU 1101 O TYR A 373 4648 4556 4281 −4 −149 −115 O ATOM 1102 N PRO A 374 8.373 4.908 −18.352 1.00 34.96 N ANISOU 1102 N PRO A 374 4537 4551 4194 45 −66 59 N ATOM 1103 CA PRO A 374 9.466 5.844 −18.520 1.00 35.26 C ANISOU 1103 CA PRO A 374 4581 4561 4251 42 −69 28 C ATOM 1104 CB PRO A 374 8.950 7.099 −17.813 1.00 35.21 C ANISOU 1104 CB PRO A 374 4553 4560 4265 58 −42 60 C ATOM 1105 CG PRO A 374 7.997 6.614 −16.837 1.00 35.05 C ANISOU 1105 CG PRO A 374 4419 4469 4428 84 −49 79 C ATOM 1106 CD PRO A 374 7.309 5.495 −17.527 1.00 35.26 C ANISOU 1106 CD PRO A 374 4566 4516 4316 65 −103 72 C ATOM 1107 C PRO A 374 10.783 5.361 −17.913 1.00 35.32 C ANISOU 1107 C PRO A 374 4595 4588 4236 62 −26 0 C ATOM 1108 O PRO A 374 10.835 4.264 −17.344 1.00 35.13 O ANISOU 1108 O PRO A 374 4500 4669 4178 31 −57 −52 O ATOM 1109 N SER A 375 11.825 6.180 −18.055 1.00 35.27 N ANISOU 1109 N SER A 375 4649 4543 4206 33 −50 −18 N ATOM 1110 CA SER A 375 13.189 5.833 −17.672 1.00 35.64 C ANISOU 1110 CA SER A 375 4661 4565 4316 56 −9 −5 C ATOM 1111 CB SER A 375 14.180 6.826 −18.293 1.00 35.47 C ANISOU 1111 CB SER A 375 4643 4554 4277 90 −42 37 C ATOM 1112 OG SER A 375 14.059 8.122 −17.713 1.00 35.13 O ANISOU 1112 OG SER A 375 4615 4635 4097 −15 −115 99 O ATOM 1113 C SER A 375 13.422 5.744 −16.151 1.00 36.11 C ANISOU 1113 C SER A 375 4725 4626 4366 77 20 5 C ATOM 1114 O SER A 375 14.398 5.153 −15.722 1.00 35.49 O ANISOU 1114 O SER A 375 4766 4553 4165 116 99 −42 O ATOM 1115 N ASP A 376 12.533 6.347 −15.358 1.00 36.79 N ANISOU 1115 N ASP A 376 4838 4675 4462 79 35 8 N ATOM 1116 CA ASP A 376 12.622 6.326 −13.893 1.00 37.26 C ANISOU 1116 CA ASP A 376 4809 4708 4637 54 59 −73 C ATOM 1117 CB ASP A 376 11.505 7.175 −13.281 1.00 37.66 C ANISOU 1117 CB ASP A 376 4923 4705 4679 39 24 −73 C ATOM 1118 CG ASP A 376 11.316 8.474 −14.010 1.00 37.98 C ANISOU 1118 CG ASP A 376 5073 4681 4676 103 74 −82 C ATOM 1119 OD1 ASP A 376 10.191 8.720 −14.495 1.00 38.29 O ANISOU 1119 OD1 ASP A 376 5022 4872 4652 110 58 −124 O ATOM 1120 OD2 ASP A 376 12.302 9.227 −14.118 1.00 35.66 O ANISOU 1120 OD2 ASP A 376 4760 4253 4535 135 89 −76 O ATOM 1121 C ASP A 376 12.549 4.905 −13.354 1.00 37.15 C ANISOU 1121 C ASP A 376 4817 4718 4579 80 84 −76 C ATOM 1122 O ASP A 376 11.569 4.191 −13.564 1.00 36.68 O ANISOU 1122 O ASP A 376 4770 4615 4551 97 140 −112 O ATOM 1123 N ILE A 377 13.600 4.508 −12.657 1.00 37.17 N ANISOU 1123 N ILE A 377 4813 4723 4588 72 69 −107 N ATOM 1124 CA ILE A 377 13.763 3.128 −12.208 1.00 37.39 C ANISOU 1124 CA ILE A 377 4797 4801 4607 45 40 −64 C ATOM 1125 CB ILE A 377 14.301 2.230 −13.385 1.00 37.15 C ANISOU 1125 CB ILE A 377 4748 4758 4609 63 21 −92 C ATOM 1126 CG1 ILE A 377 14.247 0.732 −13.024 1.00 36.97 C ANISOU 1126 CG1 ILE A 377 4634 4816 4597 20 −23 −72 C ATOM 1127 CD1 ILE A 377 14.240 −0.238 −14.232 1.00 36.57 C ANISOU 1127 CD1 ILE A 377 4633 4701 4559 59 46 −11 C ATOM 1128 CG2 ILE A 377 15.680 2.712 −13.815 1.00 35.92 C ANISOU 1128 CG2 ILE A 377 4679 4583 4385 86 48 −83 C ATOM 1129 C ILE A 377 14.714 3.107 −11.004 1.00 37.34 C ANISOU 1129 C ILE A 377 4800 4829 4556 19 37 −66 C ATOM 1130 O ILE A 377 15.420 4.080 −10.762 1.00 37.26 O ANISOU 1130 O ILE A 377 4794 4872 4491 24 40 −63 O ATOM 1131 N ALA A 378 14.715 2.004 −10.259 1.00 37.20 N ANISOU 1131 N ALA A 378 4763 4859 4511 46 40 −67 N ATOM 1132 CA ALA A 378 15.600 1.828 −9.108 1.00 37.25 C ANISOU 1132 CA ALA A 378 4784 4835 4533 7 69 −43 C ATOM 1133 CB ALA A 378 14.914 2.277 −7.836 1.00 36.48 C ANISOU 1133 CB ALA A 378 4699 4751 4411 9 47 −91 C ATOM 1134 C ALA A 378 16.010 0.362 −9.005 1.00 36.92 C ANISOU 1134 C ALA A 378 4754 4825 4447 10 95 −25 C ATOM 1135 O ALA A 378 15.193 −0.530 −9.200 1.00 36.27 O ANISOU 1135 O ALA A 378 4740 4750 4291 −5 121 −49 O ATOM 1136 N VAL A 379 17.279 0.120 −8.715 1.00 36.82 N ANISOU 1136 N VAL A 379 4725 4842 4420 2 94 1 N ATOM 1137 CA VAL A 379 17.784 −1.248 −8.628 1.00 37.12 C ANISOU 1137 CA VAL A 379 4726 4809 4568 −25 45 13 C ATOM 1138 CB VAL A 379 18.544 −1.709 −9.905 1.00 36.60 C ANISOU 1138 CB VAL A 379 4604 4751 4550 −2 47 −13 C ATOM 1139 CG1 VAL A 379 18.767 −3.219 −9.875 1.00 35.07 C ANISOU 1139 CG1 VAL A 379 4394 4622 4307 −46 −4 35 C ATOM 1140 CG2 VAL A 379 17.795 −1.317 −11.182 1.00 35.08 C ANISOU 1140 CG2 VAL A 379 4482 4481 4363 10 124 −1 C ATOM 1141 C VAL A 379 18.671 −1.420 −7.396 1.00 38.16 C ANISOU 1141 C VAL A 379 4798 4927 4773 −24 7 3 C ATOM 1142 O VAL A 379 19.429 −0.532 −7.037 1.00 38.18 O ANISOU 1142 O VAL A 379 4785 4914 4805 −16 −25 12 O ATOM 1143 N GLU A 380 18.537 −2.590 −6.782 1.00 39.23 N ANISOU 1143 N GLU A 380 4938 5049 4919 −34 −11 35 N ATOM 1144 CA GLU A 380 19.132 −2.979 −5.521 1.00 39.91 C ANISOU 1144 CA GLU A 380 5001 5151 5009 −14 −15 58 C ATOM 1145 CB GLU A 380 18.105 −2.836 −4.402 1.00 40.43 C ANISOU 1145 CB GLU A 380 5124 5190 5045 −5 −16 64 C ATOM 1146 CG GLU A 380 18.362 −1.738 −3.389 1.00 42.17 C ANISOU 1146 CG GLU A 380 5360 5320 5342 32 −171 80 C ATOM 1147 CD GLU A 380 17.614 −2.017 −2.100 1.00 42.78 C ANISOU 1147 CD GLU A 380 5158 5727 5370 116 5 121 C ATOM 1148 OE1 GLU A 380 18.231 −2.566 −1.163 1.00 47.83 O ANISOU 1148 OE1 GLU A 380 6101 6126 5945 −126 75 20 O ATOM 1149 OE2 GLU A 380 16.400 −1.757 −2.035 1.00 44.96 O ANISOU 1149 OE2 GLU A 380 5954 5476 5650 139 −62 98 O ATOM 1150 C GLU A 380 19.455 −4.455 −5.653 1.00 39.85 C ANISOU 1150 C GLU A 380 5002 5135 5004 −37 4 77 C ATOM 1151 O GLU A 380 18.764 −5.188 −6.373 1.00 40.25 O ANISOU 1151 O GLU A 380 5041 5193 5055 −78 −3 155 O ATOM 1152 N TRP A 381 20.525 −4.883 −4.991 1.00 39.62 N ANISOU 1152 N TRP A 381 4988 5103 4960 −22 19 92 N ATOM 1153 CA TRP A 381 20.811 −6.302 −4.832 1.00 39.46 C ANISOU 1153 CA TRP A 381 4896 5090 5004 38 −4 35 C ATOM 1154 CB TRP A 381 22.186 −6.672 −5.399 1.00 36.87 C ANISOU 1154 CB TRP A 381 4655 4832 4520 30 23 −91 C ATOM 1155 CG TRP A 381 22.339 −6.667 −6.907 1.00 35.55 C ANISOU 1155 CG TRP A 381 4204 4698 4603 56 −104 34 C ATOM 1156 CD1 TRP A 381 22.580 −5.579 −7.708 1.00 34.03 C ANISOU 1156 CD1 TRP A 381 4124 4434 4368 59 −71 −36 C ATOM 1157 NE1 TRP A 381 22.702 −5.972 −9.020 1.00 32.72 N ANISOU 1157 NE1 TRP A 381 4017 4085 4329 60 −50 143 N ATOM 1158 CE2 TRP A 381 22.554 −7.332 −9.093 1.00 33.85 C ANISOU 1158 CE2 TRP A 381 3981 4555 4323 111 −94 101 C ATOM 1159 CD2 TRP A 381 22.337 −7.806 −7.779 1.00 33.47 C ANISOU 1159 CD2 TRP A 381 3886 4432 4397 76 −69 127 C ATOM 1160 CE3 TRP A 381 22.170 −9.179 −7.580 1.00 32.88 C ANISOU 1160 CE3 TRP A 381 3976 4447 4068 −65 −127 39 C ATOM 1161 CZ3 TRP A 381 22.231 −10.030 −8.681 1.00 35.44 C ANISOU 1161 CZ3 TRP A 381 4009 4794 4660 101 −42 213 C ATOM 1162 CH2 TRP A 381 22.435 −9.520 −9.972 1.00 34.99 C ANISOU 1162 CH2 TRP A 381 4060 4680 4552 47 1 121 C ATOM 1163 CZ2 TRP A 381 22.611 −8.179 −10.191 1.00 33.99 C ANISOU 1163 CZ2 TRP A 381 4025 4431 4455 65 −84 182 C ATOM 1164 C TRP A 381 20.740 −6.654 −3.337 1.00 40.26 C ANISOU 1164 C TRP A 381 5035 5192 5067 47 −30 55 C ATOM 1165 O TRP A 381 20.945 −5.799 −2.475 1.00 39.66 O ANISOU 1165 O TRP A 381 4949 5140 4979 133 −91 17 O ATOM 1166 N GLU A 382 20.443 −7.914 −3.051 1.00 41.50 N ANISOU 1166 N GLU A 382 5218 5300 5247 27 −21 45 N ATOM 1167 CA GLU A 382 20.251 −8.407 −1.685 1.00 42.87 C ANISOU 1167 CA GLU A 382 5409 5466 5411 39 −22 74 C ATOM 1168 CB GLU A 382 18.793 −8.269 −1.244 1.00 43.55 C ANISOU 1168 CB GLU A 382 5498 5513 5534 40 6 64 C ATOM 1169 CG GLU A 382 18.342 −6.887 −0.771 1.00 46.25 C ANISOU 1169 CG GLU A 382 5998 5768 5807 −22 21 16 C ATOM 1170 CD GLU A 382 16.931 −6.573 −1.248 1.00 44.15 C ANISOU 1170 CD GLU A 382 5607 6334 4832 111 89 195 C ATOM 1171 OE1 GLU A 382 16.003 −6.614 −0.420 1.00 48.91 O ANISOU 1171 OE1 GLU A 382 6266 6173 6143 76 −184 −79 O ATOM 1172 OE2 GLU A 382 16.748 −6.330 −2.470 1.00 51.39 O ANISOU 1172 OE2 GLU A 382 6325 6387 6814 −32 −58 41 O ATOM 1173 C GLU A 382 20.600 −9.891 −1.653 1.00 43.58 C ANISOU 1173 C GLU A 382 5532 5524 5502 23 −11 78 C ATOM 1174 O GLU A 382 20.553 −10.579 −2.697 1.00 42.96 O ANISOU 1174 O GLU A 382 5527 5460 5335 −5 −20 53 O ATOM 1175 N SER A 383 20.946 −10.360 −0.445 1.00 44.09 N ANISOU 1175 N SER A 383 5623 5628 5499 25 18 122 N ATOM 1176 CA SER A 383 21.216 −11.777 −0.161 1.00 44.79 C ANISOU 1176 CA SER A 383 5700 5676 5639 6 37 118 C ATOM 1177 CB SER A 383 22.699 −12.113 −0.341 1.00 44.80 C ANISOU 1177 CB SER A 383 5702 5678 5642 31 37 101 C ATOM 1178 OG SER A 383 22.882 −13.478 −0.685 1.00 45.36 O ANISOU 1178 OG SER A 383 5763 5641 5831 −6 83 127 O ATOM 1179 C SER A 383 20.796 −12.028 1.283 1.00 45.26 C ANISOU 1179 C SER A 383 5775 5768 5653 −9 11 120 C ATOM 1180 O SER A 383 20.992 −11.161 2.160 1.00 45.04 O ANISOU 1180 O SER A 383 5758 5784 5569 19 43 149 O ATOM 1181 N ASN A 384 20.208 −13.202 1.518 1.00 46.24 N ANISOU 1181 N ASN A 384 5912 5856 5798 7 −6 130 N ATOM 1182 CA ASN A 384 19.541 −13.516 2.797 1.00 47.30 C ANISOU 1182 CA ASN A 384 5994 6002 5973 9 40 89 C ATOM 1183 CB ASN A 384 20.223 −14.686 3.546 1.00 47.81 C ANISOU 1183 CB ASN A 384 6076 6055 6032 50 28 85 C ATOM 1184 CG ASN A 384 21.711 −14.835 3.202 1.00 49.23 C ANISOU 1184 CG ASN A 384 6239 6244 6220 11 −19 15 C ATOM 1185 OD1 ASN A 384 22.561 −14.139 3.771 1.00 51.62 O ANISOU 1185 OD1 ASN A 384 6694 6509 6407 −81 −122 −51 O ATOM 1186 ND2 ASN A 384 22.030 −15.768 2.276 1.00 50.24 N ANISOU 1186 ND2 ASN A 384 6461 6386 6242 154 −7 90 N ATOM 1187 C ASN A 384 19.305 −12.315 3.720 1.00 47.46 C ANISOU 1187 C ASN A 384 6002 5998 6034 3 30 68 C ATOM 1188 O ASN A 384 19.988 −12.138 4.745 1.00 47.50 O ANISOU 1188 O ASN A 384 5997 6046 6002 −31 14 128 O ATOM 1189 N GLY A 385 18.354 −11.468 3.321 1.00 48.01 N ANISOU 1189 N GLY A 385 6027 6061 6150 −4 39 51 N ATOM 1190 CA GLY A 385 17.871 −10.396 4.198 1.00 48.23 C ANISOU 1190 CA GLY A 385 6064 6061 6200 13 51 19 C ATOM 1191 C GLY A 385 18.621 −9.098 4.087 1.00 48.46 C ANISOU 1191 C GLY A 385 6080 6127 6205 −3 47 15 C ATOM 1192 O GLY A 385 18.014 −8.041 3.849 1.00 49.13 O ANISOU 1192 O GLY A 385 6172 6193 6300 0 74 −4 O ATOM 1193 N GLN A 386 19.938 −9.186 4.258 1.00 48.32 N ANISOU 1193 N GLN A 386 6069 6151 6136 −3 −15 14 N ATOM 1194 CA GLN A 386 20.841 −8.054 4.091 1.00 48.32 C ANISOU 1194 CA GLN A 386 6053 6229 6077 −28 −15 30 C ATOM 1195 CB GLN A 386 22.184 −8.360 4.790 1.00 48.91 C ANISOU 1195 CB GLN A 386 6138 6313 6130 −23 −19 49 C ATOM 1196 CG GLN A 386 22.173 −8.184 6.332 1.00 49.93 C ANISOU 1196 CG GLN A 386 6349 6323 6297 12 17 −18 C ATOM 1197 CD GLN A 386 21.542 −6.872 6.784 1.00 53.93 C ANISOU 1197 CD GLN A 386 7210 6843 6437 62 21 77 C ATOM 1198 OE1 GLN A 386 22.249 −5.898 7.064 1.00 52.63 O ANISOU 1198 OE1 GLN A 386 6586 6564 6846 −191 −29 −6 O ATOM 1199 NE2 GLN A 386 20.209 −6.839 6.856 1.00 50.58 N ANISOU 1199 NE2 GLN A 386 6207 6607 6405 −46 59 43 N ATOM 1200 C GLN A 386 21.064 −7.587 2.620 1.00 47.59 C ANISOU 1200 C GLN A 386 5946 6111 6025 −11 −32 25 C ATOM 1201 O GLN A 386 20.871 −8.355 1.660 1.00 47.43 O ANISOU 1201 O GLN A 386 5872 6171 5978 −14 5 −8 O ATOM 1202 N PRO A 387 21.469 −6.317 2.452 1.00 46.79 N ANISOU 1202 N PRO A 387 5884 5981 5912 8 −30 38 N ATOM 1203 CA PRO A 387 21.779 −5.739 1.158 1.00 46.49 C ANISOU 1203 CA PRO A 387 5862 5936 5864 6 −19 30 C ATOM 1204 CB PRO A 387 21.498 −4.243 1.368 1.00 46.62 C ANISOU 1204 CB PRO A 387 5909 5918 5886 −16 −15 26 C ATOM 1205 CG PRO A 387 21.249 −4.046 2.854 1.00 46.58 C ANISOU 1205 CG PRO A 387 5910 5950 5836 −20 −43 57 C ATOM 1206 CD PRO A 387 21.622 −5.325 3.529 1.00 47.03 C ANISOU 1206 CD PRO A 387 5913 5996 5958 15 −45 16 C ATOM 1207 C PRO A 387 23.235 −5.928 0.705 1.00 46.20 C ANISOU 1207 C PRO A 387 5849 5892 5812 −33 −23 39 C ATOM 1208 O PRO A 387 24.173 −5.617 1.452 1.00 46.11 O ANISOU 1208 O PRO A 387 5879 5882 5758 −50 −52 64 O ATOM 1209 N GLU A 388 23.408 −6.433 −0.517 1.00 45.47 N ANISOU 1209 N GLU A 388 5756 5801 5720 −47 −20 52 N ATOM 1210 CA GLU A 388 24.694 −6.403 −1.195 1.00 44.97 C ANISOU 1210 CA GLU A 388 5683 5738 5662 −18 −10 44 C ATOM 1211 CB GLU A 388 24.706 −7.402 −2.338 1.00 44.97 C ANISOU 1211 CB GLU A 388 5665 5761 5658 −20 −28 47 C ATOM 1212 CG GLU A 388 24.340 −8.830 −1.945 1.00 46.39 C ANISOU 1212 CG GLU A 388 5861 5872 5891 −63 −42 82 C ATOM 1213 CD GLU A 388 25.323 −9.460 −0.970 1.00 48.03 C ANISOU 1213 CD GLU A 388 6158 6071 6019 −44 −74 23 C ATOM 1214 OE1 GLU A 388 26.552 −9.400 −1.201 1.00 47.49 O ANISOU 1214 OE1 GLU A 388 6152 6000 5891 0 39 1 O ATOM 1215 OE2 GLU A 388 24.856 −10.029 0.035 1.00 49.67 O ANISOU 1215 OE2 GLU A 388 6394 6329 6149 −70 29 56 O ATOM 1216 C GLU A 388 24.879 −4.998 −1.738 1.00 44.67 C ANISOU 1216 C GLU A 388 5627 5760 5582 −57 10 43 C ATOM 1217 O GLU A 388 24.084 −4.530 −2.545 1.00 45.07 O ANISOU 1217 O GLU A 388 5666 5799 5660 −63 32 78 O ATOM 1218 N ASN A 389 25.901 −4.298 −1.279 1.00 44.47 N ANISOU 1218 N ASN A 389 5659 5699 5537 −59 24 35 N ATOM 1219 CA ASN A 389 26.079 −2.904 −1.704 1.00 44.04 C ANISOU 1219 CA ASN A 389 5629 5641 5462 −27 10 35 C ATOM 1220 CB ASN A 389 26.024 −1.961 −0.492 1.00 44.59 C ANISOU 1220 CB ASN A 389 5722 5687 5532 −8 38 20 C ATOM 1221 CG ASN A 389 24.585 −1.678 −0.037 1.00 46.42 C ANISOU 1221 CG ASN A 389 5840 6045 5752 −29 −7 33 C ATOM 1222 OD1 ASN A 389 23.682 −1.555 −0.863 1.00 47.34 O ANISOU 1222 OD1 ASN A 389 6045 6314 5625 −18 −114 −3 O ATOM 1223 ND2 ASN A 389 24.374 −1.565 1.288 1.00 48.67 N ANISOU 1223 ND2 ASN A 389 6260 6364 5865 −48 −31 −51 N ATOM 1224 C ASN A 389 27.307 −2.672 −2.610 1.00 43.16 C ANISOU 1224 C ASN A 389 5533 5513 5352 −26 18 33 C ATOM 1225 O ASN A 389 27.627 −1.534 −2.972 1.00 43.60 O ANISOU 1225 O ASN A 389 5592 5599 5374 −86 14 23 O ATOM 1226 N ASN A 390 27.945 −3.773 −2.997 1.00 41.86 N ANISOU 1226 N ASN A 390 5352 5379 5173 23 45 56 N ATOM 1227 CA ASN A 390 29.083 −3.793 −3.919 1.00 41.00 C ANISOU 1227 CA ASN A 390 5220 5249 5109 20 7 74 C ATOM 1228 CB ASN A 390 29.966 −4.979 −3.549 1.00 41.19 C ANISOU 1228 CB ASN A 390 5221 5310 5120 45 27 70 C ATOM 1229 CG ASN A 390 31.368 −4.870 −4.090 1.00 41.56 C ANISOU 1229 CG ASN A 390 5312 5363 5115 −2 43 34 C ATOM 1230 OD1 ASN A 390 31.868 −3.776 −4.364 1.00 41.19 O ANISOU 1230 OD1 ASN A 390 5395 5253 5003 −42 153 41 O ATOM 1231 ND2 ASN A 390 32.025 −6.025 −4.233 1.00 41.17 N ANISOU 1231 ND2 ASN A 390 5229 5437 4976 143 37 31 N ATOM 1232 C ASN A 390 28.594 −3.918 −5.382 1.00 40.10 C ANISOU 1232 C ASN A 390 5080 5147 5009 44 50 76 C ATOM 1233 O ASN A 390 28.961 −4.838 −6.130 1.00 39.50 O ANISOU 1233 O ASN A 390 5007 5028 4970 3 76 100 O ATOM 1234 N TYR A 391 27.732 −2.989 −5.776 1.00 38.85 N ANISOU 1234 N TYR A 391 4892 5023 4845 30 31 97 N ATOM 1235 CA TYR A 391 27.123 −3.060 −7.093 1.00 37.67 C ANISOU 1235 CA TYR A 391 4723 4859 4729 39 55 30 C ATOM 1236 CB TYR A 391 25.674 −3.607 −7.023 1.00 37.59 C ANISOU 1236 CB TYR A 391 4770 4816 4693 80 13 10 C ATOM 1237 CG TYR A 391 24.671 −2.674 −6.375 1.00 38.73 C ANISOU 1237 CG TYR A 391 4872 5036 4808 −63 −19 82 C ATOM 1238 CD1 TYR A 391 24.362 −2.780 −5.014 1.00 37.63 C ANISOU 1238 CD1 TYR A 391 4713 4862 4722 177 116 46 C ATOM 1239 CE1 TYR A 391 23.443 −1.907 −4.407 1.00 38.01 C ANISOU 1239 CE1 TYR A 391 4865 4872 4702 −18 48 130 C ATOM 1240 CZ TYR A 391 22.846 −0.928 −5.185 1.00 38.97 C ANISOU 1240 CZ TYR A 391 4955 4954 4896 −4 −26 90 C ATOM 1241 OH TYR A 391 21.946 −0.056 −4.628 1.00 37.74 O ANISOU 1241 OH TYR A 391 4898 4791 4649 209 262 −75 O ATOM 1242 CE2 TYR A 391 23.138 −0.815 −6.539 1.00 35.97 C ANISOU 1242 CE2 TYR A 391 4646 4399 4619 38 163 83 C ATOM 1243 CD2 TYR A 391 24.039 −1.671 −7.119 1.00 36.37 C ANISOU 1243 CD2 TYR A 391 4651 4617 4551 −25 77 44 C ATOM 1244 C TYR A 391 27.200 −1.701 −7.765 1.00 36.92 C ANISOU 1244 C TYR A 391 4614 4817 4594 42 51 55 C ATOM 1245 O TYR A 391 27.351 −0.657 −7.114 1.00 36.53 O ANISOU 1245 O TYR A 391 4501 4815 4562 −50 −10 88 O ATOM 1246 N LYS A 392 27.118 −1.716 −9.081 1.00 36.62 N ANISOU 1246 N LYS A 392 4561 4727 4624 54 58 1 N ATOM 1247 CA LYS A 392 27.049 −0.484 −9.842 1.00 35.22 C ANISOU 1247 CA LYS A 392 4471 4551 4359 25 92 57 C ATOM 1248 CB LYS A 392 28.381 −0.165 −10.507 1.00 34.96 C ANISOU 1248 CB LYS A 392 4479 4541 4263 56 38 59 C ATOM 1249 CG LYS A 392 29.467 0.393 −9.610 1.00 33.97 C ANISOU 1249 CG LYS A 392 4364 4380 4162 9 118 142 C ATOM 1250 CD LYS A 392 29.178 1.783 −9.077 1.00 30.65 C ANISOU 1250 CD LYS A 392 3873 4093 3676 −50 −59 22 C ATOM 1251 CE LYS A 392 30.097 2.063 −7.905 1.00 34.02 C ANISOU 1251 CE LYS A 392 4211 4391 4322 54 232 178 C ATOM 1252 NZ LYS A 392 29.956 3.443 −7.308 1.00 33.12 N ANISOU 1252 NZ LYS A 392 4344 4095 4143 80 8 56 N ATOM 1253 C LYS A 392 25.990 −0.715 −10.856 1.00 34.87 C ANISOU 1253 C LYS A 392 4403 4536 4310 11 107 58 C ATOM 1254 O LYS A 392 25.872 −1.819 −11.377 1.00 35.62 O ANISOU 1254 O LYS A 392 4423 4675 4433 32 181 150 O ATOM 1255 N THR A 393 25.182 0.301 −11.113 1.00 34.71 N ANISOU 1255 N THR A 393 4367 4517 4301 −7 76 10 N ATOM 1256 CA THR A 393 24.148 0.202 −12.152 1.00 34.16 C ANISOU 1256 CA THR A 393 4265 4432 4281 0 19 3 C ATOM 1257 CB THR A 393 22.709 0.335 −11.564 1.00 34.10 C ANISOU 1257 CB THR A 393 4267 4421 4266 −9 30 −26 C ATOM 1258 OG1 THR A 393 22.539 −0.610 −10.500 1.00 33.81 O ANISOU 1258 OG1 THR A 393 4259 4412 4172 −1 −82 −7 O ATOM 1259 CG2 THR A 393 21.632 0.074 −12.619 1.00 32.60 C ANISOU 1259 CG2 THR A 393 4085 4261 4038 42 66 47 C ATOM 1260 C THR A 393 24.399 1.248 −13.221 1.00 33.43 C ANISOU 1260 C THR A 393 4164 4382 4153 14 6 11 C ATOM 1261 O THR A 393 24.712 2.402 −12.916 1.00 32.42 O ANISOU 1261 O THR A 393 3933 4348 4036 27 −28 52 O ATOM 1262 N THR A 394 24.280 0.831 −14.475 1.00 33.41 N ANISOU 1262 N THR A 394 4194 4326 4173 −21 1 −3 N ATOM 1263 CA THR A 394 24.354 1.770 −15.604 1.00 32.96 C ANISOU 1263 CA THR A 394 4273 4227 4022 −7 8 −20 C ATOM 1264 CB THR A 394 24.438 1.072 −17.010 1.00 32.13 C ANISOU 1264 CB THR A 394 4197 4083 3928 −5 10 −4 C ATOM 1265 OG1 THR A 394 23.164 0.562 −17.403 1.00 30.15 O ANISOU 1265 OG1 THR A 394 4301 3693 3462 100 189 −92 O ATOM 1266 CG2 THR A 394 25.420 −0.023 −17.015 1.00 32.17 C ANISOU 1266 CG2 THR A 394 4155 4105 3960 10 −1 −49 C ATOM 1267 C THR A 394 23.180 2.745 −15.578 1.00 33.10 C ANISOU 1267 C THR A 394 4317 4262 3996 22 −31 −57 C ATOM 1268 O THR A 394 22.111 2.399 −15.084 1.00 31.47 O ANISOU 1268 O THR A 394 4242 4074 3640 41 −14 −63 O ATOM 1269 N PRO A 395 23.384 3.974 −16.096 1.00 33.99 N ANISOU 1269 N PRO A 395 4452 4367 4095 17 0 −46 N ATOM 1270 CA PRO A 395 22.219 4.810 −16.436 1.00 34.48 C ANISOU 1270 CA PRO A 395 4508 4400 4191 5 0 −2 C ATOM 1271 CB PRO A 395 22.838 6.025 −17.138 1.00 34.62 C ANISOU 1271 CB PRO A 395 4510 4435 4207 39 10 16 C ATOM 1272 CG PRO A 395 24.252 6.053 −16.758 1.00 34.22 C ANISOU 1272 CG PRO A 395 4486 4452 4064 2 −63 −21 C ATOM 1273 CD PRO A 395 24.661 4.648 −16.377 1.00 33.86 C ANISOU 1273 CD PRO A 395 4399 4344 4121 12 −25 −108 C ATOM 1274 C PRO A 395 21.270 4.066 −17.399 1.00 34.85 C ANISOU 1274 C PRO A 395 4538 4450 4251 −27 24 75 C ATOM 1275 O PRO A 395 21.690 3.069 −18.027 1.00 33.16 O ANISOU 1275 O PRO A 395 4370 4274 3952 −56 11 94 O ATOM 1276 N PRO A 396 19.994 4.510 −17.489 1.00 35.07 N ANISOU 1276 N PRO A 396 4573 4414 4338 −15 31 90 N ATOM 1277 CA PRO A 396 19.125 3.923 −18.487 1.00 35.48 C ANISOU 1277 CA PRO A 396 4599 4477 4402 −4 21 48 C ATOM 1278 CB PRO A 396 17.743 4.529 −18.186 1.00 35.53 C ANISOU 1278 CB PRO A 396 4659 4433 4407 −25 75 67 C ATOM 1279 CG PRO A 396 17.867 5.217 −16.873 1.00 35.18 C ANISOU 1279 CG PRO A 396 4624 4408 4334 −59 3 93 C ATOM 1280 CD PRO A 396 19.303 5.531 −16.681 1.00 35.32 C ANISOU 1280 CD PRO A 396 4594 4469 4356 −36 28 82 C ATOM 1281 C PRO A 396 19.586 4.324 −19.886 1.00 35.60 C ANISOU 1281 C PRO A 396 4558 4488 4477 −4 25 45 C ATOM 1282 O PRO A 396 19.954 5.475 −20.105 1.00 34.99 O ANISOU 1282 O PRO A 396 4438 4447 4409 −29 −6 −1 O ATOM 1283 N VAL A 397 19.574 3.361 −20.811 1.00 35.51 N ANISOU 1283 N VAL A 397 4544 4506 4441 17 17 29 N ATOM 1284 CA VAL A 397 19.934 3.603 −22.199 1.00 35.28 C ANISOU 1284 CA VAL A 397 4527 4479 4397 49 41 −32 C ATOM 1285 CB VAL A 397 21.078 2.664 −22.649 1.00 35.15 C ANISOU 1285 CB VAL A 397 4516 4432 4406 15 −5 15 C ATOM 1286 CG1 VAL A 397 21.631 3.022 −24.077 1.00 32.51 C ANISOU 1286 CG1 VAL A 397 4128 4063 4159 162 −111 −9 C ATOM 1287 CG2 VAL A 397 22.212 2.715 −21.598 1.00 34.48 C ANISOU 1287 CG2 VAL A 397 4490 4344 4264 57 −19 107 C ATOM 1288 C VAL A 397 18.679 3.500 −23.052 1.00 35.75 C ANISOU 1288 C VAL A 397 4638 4562 4383 61 96 −101 C ATOM 1289 O VAL A 397 17.902 2.554 −22.928 1.00 35.03 O ANISOU 1289 O VAL A 397 4596 4513 4200 112 126 −186 O ATOM 1290 N LEU A 398 18.454 4.511 −23.882 1.00 36.47 N ANISOU 1290 N LEU A 398 4739 4629 4488 94 73 −100 N ATOM 1291 CA LEU A 398 17.325 4.490 −24.793 1.00 37.45 C ANISOU 1291 CA LEU A 398 4793 4772 4664 41 20 −48 C ATOM 1292 CB LEU A 398 17.122 5.874 −25.398 1.00 37.18 C ANISOU 1292 CB LEU A 398 4747 4739 4639 16 −9 −44 C ATOM 1293 CG LEU A 398 16.080 5.972 −26.506 1.00 37.09 C ANISOU 1293 CG LEU A 398 4734 4681 4674 50 −12 25 C ATOM 1294 CD1 LEU A 398 14.703 6.031 −25.905 1.00 33.79 C ANISOU 1294 CD1 LEU A 398 4353 4255 4230 41 −3 61 C ATOM 1295 CD2 LEU A 398 16.370 7.193 −27.347 1.00 35.57 C ANISOU 1295 CD2 LEU A 398 4571 4494 4446 −15 49 72 C ATOM 1296 C LEU A 398 17.556 3.429 −25.888 1.00 38.23 C ANISOU 1296 C LEU A 398 4890 4908 4728 11 15 −44 C ATOM 1297 O LEU A 398 18.563 3.460 −26.582 1.00 38.51 O ANISOU 1297 O LEU A 398 4999 4941 4690 0 55 −39 O ATOM 1298 N ASP A 399 16.635 2.480 −25.991 1.00 38.60 N ANISOU 1298 N ASP A 399 4899 4972 4792 −36 −27 −45 N ATOM 1299 CA ASP A 399 16.765 1.320 −26.873 1.00 39.01 C ANISOU 1299 CA ASP A 399 4912 5053 4854 −29 24 −35 C ATOM 1300 CB ASP A 399 16.199 0.086 −26.161 1.00 38.88 C ANISOU 1300 CB ASP A 399 4905 4978 4887 15 −41 −51 C ATOM 1301 CG ASP A 399 16.844 −1.192 −26.597 1.00 38.32 C ANISOU 1301 CG ASP A 399 4804 4963 4792 −65 −106 −121 C ATOM 1302 OD1 ASP A 399 17.484 −1.181 −27.654 1.00 37.78 O ANISOU 1302 OD1 ASP A 399 4733 4944 4677 −131 237 20 O ATOM 1303 OD2 ASP A 399 16.695 −2.218 −25.888 1.00 34.88 O ANISOU 1303 OD2 ASP A 399 4527 4630 4095 −144 −197 −160 O ATOM 1304 C ASP A 399 16.021 1.580 −28.189 1.00 39.56 C ANISOU 1304 C ASP A 399 4970 5121 4940 −44 −39 −55 C ATOM 1305 O ASP A 399 15.173 2.463 −28.256 1.00 39.73 O ANISOU 1305 O ASP A 399 4986 5183 4925 −115 −74 −71 O ATOM 1306 N SER A 400 16.337 0.812 −29.230 1.00 40.64 N ANISOU 1306 N SER A 400 5096 5273 5071 −57 −46 −54 N ATOM 1307 CA SER A 400 15.840 1.082 −30.596 1.00 41.15 C ANISOU 1307 CA SER A 400 5251 5323 5061 −17 −32 −8 C ATOM 1308 CB SER A 400 16.537 0.177 −31.634 1.00 41.51 C ANISOU 1308 CB SER A 400 5316 5343 5111 −10 −34 −7 C ATOM 1309 OG SER A 400 16.267 −1.193 −31.421 1.00 41.13 O ANISOU 1309 OG SER A 400 5528 5227 4871 −68 −46 −78 O ATOM 1310 C SER A 400 14.306 1.094 −30.795 1.00 41.37 C ANISOU 1310 C SER A 400 5252 5362 5103 −45 −34 −4 C ATOM 1311 O SER A 400 13.809 1.646 −31.793 1.00 41.91 O ANISOU 1311 O SER A 400 5272 5451 5198 −42 −44 15 O ATOM 1312 N ASP A 401 13.567 0.507 −29.850 1.00 40.91 N ANISOU 1312 N ASP A 401 5201 5318 5025 −65 −57 −11 N ATOM 1313 CA ASP A 401 12.096 0.529 −29.865 1.00 40.44 C ANISOU 1313 CA ASP A 401 5132 5251 4983 −27 −42 18 C ATOM 1314 CB ASP A 401 11.534 −0.798 −29.335 1.00 40.38 C ANISOU 1314 CB ASP A 401 5139 5233 4968 −25 −21 −7 C ATOM 1315 CG ASP A 401 11.904 −1.060 −27.886 1.00 38.85 C ANISOU 1315 CG ASP A 401 4987 4862 4910 −73 −13 26 C ATOM 1316 OD1 ASP A 401 12.436 −0.155 −27.202 1.00 37.74 O ANISOU 1316 OD1 ASP A 401 4636 5156 4545 38 31 174 O ATOM 1317 OD2 ASP A 401 11.649 −2.183 −27.431 1.00 39.72 O ANISOU 1317 OD2 ASP A 401 5010 5104 4977 −4 75 7 O ATOM 1318 C ASP A 401 11.449 1.680 −29.094 1.00 39.90 C ANISOU 1318 C ASP A 401 5041 5177 4940 −43 −37 66 C ATOM 1319 O ASP A 401 10.225 1.774 −29.043 1.00 40.25 O ANISOU 1319 O ASP A 401 5049 5289 4953 −69 −45 118 O ATOM 1320 N GLY A 402 12.250 2.543 −28.477 1.00 39.08 N ANISOU 1320 N GLY A 402 4955 5065 4828 −36 −20 89 N ATOM 1321 CA GLY A 402 11.704 3.649 −27.704 1.00 37.86 C ANISOU 1321 CA GLY A 402 4786 4893 4704 −52 −13 78 C ATOM 1322 C GLY A 402 11.595 3.341 −26.218 1.00 37.33 C ANISOU 1322 C GLY A 402 4692 4826 4663 −63 −17 32 C ATOM 1323 O GLY A 402 11.207 4.196 −25.442 1.00 36.37 O ANISOU 1323 O GLY A 402 4563 4782 4474 −59 −1 82 O ATOM 1324 N SER A 403 11.926 2.109 −25.833 1.00 37.09 N ANISOU 1324 N SER A 403 4673 4844 4574 −62 −36 47 N ATOM 1325 CA SER A 403 11.950 1.716 −24.434 1.00 36.37 C ANISOU 1325 CA SER A 403 4631 4747 4437 −49 −21 −12 C ATOM 1326 CB SER A 403 11.448 0.278 −24.256 1.00 36.02 C ANISOU 1326 CB SER A 403 4574 4762 4349 −7 −46 2 C ATOM 1327 OG SER A 403 12.414 −0.692 −24.631 1.00 33.78 O ANISOU 1327 OG SER A 403 4447 4556 3833 −138 −49 0 O ATOM 1328 C SER A 403 13.386 1.883 −23.941 1.00 36.54 C ANISOU 1328 C SER A 403 4660 4740 4482 −12 25 −5 C ATOM 1329 O SER A 403 14.295 2.067 −24.761 1.00 36.38 O ANISOU 1329 O SER A 403 4672 4720 4430 −40 37 14 O ATOM 1330 N PHE A 404 13.581 1.845 −22.620 1.00 35.39 N ANISOU 1330 N PHE A 404 4542 4621 4282 −38 41 −73 N ATOM 1331 CA PHE A 404 14.907 1.883 −22.036 1.00 34.51 C ANISOU 1331 CA PHE A 404 4526 4498 4089 6 74 −104 C ATOM 1332 CB PHE A 404 14.966 2.842 −20.820 1.00 33.40 C ANISOU 1332 CB PHE A 404 4419 4290 3980 33 38 −95 C ATOM 1333 CG PHE A 404 14.744 4.276 −21.166 1.00 30.44 C ANISOU 1333 CG PHE A 404 4161 4147 3255 −31 −6 −191 C ATOM 1334 CD1 PHE A 404 15.823 5.101 −21.466 1.00 26.60 C ANISOU 1334 CD1 PHE A 404 3621 3887 2596 99 −79 −402 C ATOM 1335 CE1 PHE A 404 15.644 6.447 −21.816 1.00 26.39 C ANISOU 1335 CE1 PHE A 404 3482 3882 2662 −15 8 −185 C ATOM 1336 CZ PHE A 404 14.347 6.978 −21.858 1.00 32.27 C ANISOU 1336 CZ PHE A 404 4351 4333 3573 50 −33 −132 C ATOM 1337 CE2 PHE A 404 13.225 6.145 −21.550 1.00 29.16 C ANISOU 1337 CE2 PHE A 404 3933 3731 3415 −11 40 −51 C ATOM 1338 CD2 PHE A 404 13.445 4.805 −21.208 1.00 30.50 C ANISOU 1338 CD2 PHE A 404 4166 4173 3248 −128 106 −201 C ATOM 1339 C PHE A 404 15.373 0.499 −21.608 1.00 34.61 C ANISOU 1339 C PHE A 404 4525 4477 4146 26 99 −204 C ATOM 1340 O PHE A 404 14.573 −0.420 −21.433 1.00 33.02 O ANISOU 1340 O PHE A 404 4344 4311 3889 47 220 −276 O ATOM 1341 N PHE A 405 16.690 0.368 −21.449 1.00 34.74 N ANISOU 1341 N PHE A 405 4575 4438 4184 43 52 −212 N ATOM 1342 CA PHE A 405 17.272 −0.759 −20.737 1.00 34.42 C ANISOU 1342 CA PHE A 405 4502 4382 4192 −2 44 −125 C ATOM 1343 CB PHE A 405 17.776 −1.837 −21.701 1.00 32.43 C ANISOU 1343 CB PHE A 405 4151 4159 4010 −72 155 −97 C ATOM 1344 CG PHE A 405 19.014 −1.437 −22.476 1.00 33.02 C ANISOU 1344 CG PHE A 405 4397 3928 4220 8 −32 −287 C ATOM 1345 CD1 PHE A 405 20.277 −1.889 −22.081 1.00 29.57 C ANISOU 1345 CD1 PHE A 405 4067 3437 3730 −38 −280 −316 C ATOM 1346 CE1 PHE A 405 21.400 −1.541 −22.787 1.00 25.41 C ANISOU 1346 CE1 PHE A 405 3611 3087 2955 −141 −47 125 C ATOM 1347 CZ PHE A 405 21.292 −0.716 −23.897 1.00 32.01 C ANISOU 1347 CZ PHE A 405 4279 3768 4114 −148 −68 −437 C ATOM 1348 CE2 PHE A 405 20.048 −0.245 −24.310 1.00 27.60 C ANISOU 1348 CE2 PHE A 405 3778 3209 3499 −32 −193 −158 C ATOM 1349 CD2 PHE A 405 18.915 −0.607 −23.606 1.00 29.13 C ANISOU 1349 CD2 PHE A 405 4022 3536 3508 −50 −6 −170 C ATOM 1350 C PHE A 405 18.415 −0.275 −19.858 1.00 34.59 C ANISOU 1350 C PHE A 405 4492 4419 4230 −18 50 −131 C ATOM 1351 O PHE A 405 18.909 0.844 −20.028 1.00 33.97 O ANISOU 1351 O PHE A 405 4478 4384 4045 −56 30 −88 O ATOM 1352 N LEU A 406 18.811 −1.132 −18.922 1.00 34.15 N ANISOU 1352 N LEU A 406 4406 4368 4199 −66 89 −89 N ATOM 1353 CA LEU A 406 20.063 −0.990 −18.217 1.00 34.71 C ANISOU 1353 CA LEU A 406 4441 4465 4282 −10 69 −40 C ATOM 1354 CB LEU A 406 19.973 0.066 −17.112 1.00 34.26 C ANISOU 1354 CB LEU A 406 4416 4403 4198 0 3 −2 C ATOM 1355 CG LEU A 406 19.362 −0.080 −15.723 1.00 32.70 C ANISOU 1355 CG LEU A 406 4113 4273 4036 −57 −11 1 C ATOM 1356 CD1 LEU A 406 18.140 0.758 −15.580 1.00 31.62 C ANISOU 1356 CD1 LEU A 406 3956 4197 3860 −42 −51 147 C ATOM 1357 CD2 LEU A 406 19.149 −1.480 −15.240 1.00 30.79 C ANISOU 1357 CD2 LEU A 406 3828 4056 3815 −202 −43 −257 C ATOM 1358 C LEU A 406 20.593 −2.310 −17.687 1.00 35.03 C ANISOU 1358 C LEU A 406 4496 4493 4320 −22 88 −40 C ATOM 1359 O LEU A 406 19.908 −3.316 −17.763 1.00 35.15 O ANISOU 1359 O LEU A 406 4419 4618 4318 −7 134 −19 O ATOM 1360 N TYR A 407 21.835 −2.297 −17.198 1.00 35.31 N ANISOU 1360 N TYR A 407 4535 4528 4351 −7 40 −34 N ATOM 1361 CA TYR A 407 22.410 −3.423 −16.471 1.00 35.65 C ANISOU 1361 CA TYR A 407 4544 4568 4430 −6 40 −15 C ATOM 1362 CB TYR A 407 23.581 −4.065 −17.216 1.00 34.84 C ANISOU 1362 CB TYR A 407 4429 4478 4327 −14 12 −49 C ATOM 1363 CG TYR A 407 23.321 −4.723 −18.548 1.00 34.08 C ANISOU 1363 CG TYR A 407 4249 4291 4407 40 57 9 C ATOM 1364 CD1 TYR A 407 23.139 −3.959 −19.694 1.00 32.26 C ANISOU 1364 CD1 TYR A 407 3966 3991 4300 71 69 −29 C ATOM 1365 CE1 TYR A 407 22.945 −4.551 −20.936 1.00 31.35 C ANISOU 1365 CE1 TYR A 407 3804 3854 4251 1 −7 29 C ATOM 1366 CZ TYR A 407 22.964 −5.923 −21.046 1.00 32.84 C ANISOU 1366 CZ TYR A 407 3810 4228 4440 0 45 0 C ATOM 1367 OH TYR A 407 22.771 −6.460 −22.284 1.00 34.03 O ANISOU 1367 OH TYR A 407 4198 4350 4380 16 61 8 O ATOM 1368 CE2 TYR A 407 23.163 −6.726 −19.925 1.00 31.27 C ANISOU 1368 CE2 TYR A 407 3774 3877 4229 25 90 −36 C ATOM 1369 CD2 TYR A 407 23.354 −6.119 −18.681 1.00 32.21 C ANISOU 1369 CD2 TYR A 407 3878 4149 4210 −57 57 56 C ATOM 1370 C TYR A 407 22.935 −2.988 −15.099 1.00 35.88 C ANISOU 1370 C TYR A 407 4601 4620 4411 −27 26 −8 C ATOM 1371 O TYR A 407 23.460 −1.890 −14.934 1.00 35.29 O ANISOU 1371 O TYR A 407 4616 4570 4220 18 −14 −3 O ATOM 1372 N SER A 408 22.794 −3.888 −14.134 1.00 36.43 N ANISOU 1372 N SER A 408 4686 4700 4454 −17 21 8 N ATOM 1373 CA SER A 408 23.367 −3.750 −12.801 1.00 36.52 C ANISOU 1373 CA SER A 408 4655 4722 4499 −22 28 33 C ATOM 1374 CB SER A 408 22.296 −3.911 −11.702 1.00 36.58 C ANISOU 1374 CB SER A 408 4665 4728 4507 −32 27 15 C ATOM 1375 OG SER A 408 22.782 −3.508 −10.426 1.00 34.60 O ANISOU 1375 OG SER A 408 4359 4564 4222 70 111 165 O ATOM 1376 C SER A 408 24.397 −4.849 −12.686 1.00 36.40 C ANISOU 1376 C SER A 408 4657 4704 4467 −12 38 50 C ATOM 1377 O SER A 408 24.159 −5.985 −13.121 1.00 36.21 O ANISOU 1377 O SER A 408 4674 4649 4435 −17 72 98 O ATOM 1378 N LYS A 409 25.553 −4.484 −12.145 1.00 36.10 N ANISOU 1378 N LYS A 409 4657 4695 4362 4 −34 66 N ATOM 1379 CA LYS A 409 26.659 −5.409 −11.944 1.00 35.94 C ANISOU 1379 CA LYS A 409 4630 4629 4395 7 −40 60 C ATOM 1380 CB LYS A 409 27.906 −4.935 −12.709 1.00 35.47 C ANISOU 1380 CB LYS A 409 4588 4563 4326 −11 −12 65 C ATOM 1381 CG LYS A 409 29.104 −5.896 −12.600 1.00 36.03 C ANISOU 1381 CG LYS A 409 4596 4593 4500 −14 25 105 C ATOM 1382 CD LYS A 409 30.197 −5.695 −13.673 1.00 36.06 C ANISOU 1382 CD LYS A 409 4630 4581 4491 54 −44 108 C ATOM 1383 CE LYS A 409 31.116 −4.491 −13.396 1.00 33.48 C ANISOU 1383 CE LYS A 409 4373 4343 4003 26 −214 0 C ATOM 1384 NZ LYS A 409 31.618 −4.426 −11.979 1.00 34.29 N ANISOU 1384 NZ LYS A 409 4556 4113 4356 53 22 193 N ATOM 1385 C LYS A 409 26.941 −5.580 −10.425 1.00 35.82 C ANISOU 1385 C LYS A 409 4612 4607 4388 −8 −73 104 C ATOM 1386 O LYS A 409 27.346 −4.637 −9.738 1.00 35.11 O ANISOU 1386 O LYS A 409 4621 4589 4130 −13 −162 98 O ATOM 1387 N LEU A 410 26.673 −6.777 −9.920 1.00 35.61 N ANISOU 1387 N LEU A 410 4529 4579 4420 14 −95 141 N ATOM 1388 CA LEU A 410 26.958 −7.114 −8.531 1.00 35.76 C ANISOU 1388 CA LEU A 410 4598 4561 4426 4 1 114 C ATOM 1389 CB LEU A 410 25.857 −7.992 −7.911 1.00 35.47 C ANISOU 1389 CB LEU A 410 4524 4542 4409 −3 −25 72 C ATOM 1390 CG LEU A 410 26.145 −8.413 −6.445 1.00 35.65 C ANISOU 1390 CG LEU A 410 4571 4520 4452 10 25 122 C ATOM 1391 CD1 LEU A 410 26.011 −7.260 −5.456 1.00 33.68 C ANISOU 1391 CD1 LEU A 410 4444 4388 3963 51 76 155 C ATOM 1392 CD2 LEU A 410 25.311 −9.570 −6.007 1.00 35.81 C ANISOU 1392 CD2 LEU A 410 4430 4679 4494 −71 30 184 C ATOM 1393 C LEU A 410 28.292 −7.843 −8.495 1.00 35.82 C ANISOU 1393 C LEU A 410 4590 4560 4458 39 −21 187 C ATOM 1394 O LEU A 410 28.479 −8.850 −9.213 1.00 35.64 O ANISOU 1394 O LEU A 410 4599 4489 4455 42 −33 257 O ATOM 1395 N THR A 411 29.225 −7.315 −7.707 1.00 36.06 N ANISOU 1395 N THR A 411 4693 4596 4410 41 −16 193 N ATOM 1396 CA THR A 411 30.513 −7.989 −7.514 1.00 37.46 C ANISOU 1396 CA THR A 411 4777 4828 4625 24 0 147 C ATOM 1397 CB THR A 411 31.705 −7.010 −7.613 1.00 37.32 C ANISOU 1397 CB THR A 411 4746 4809 4625 10 −29 154 C ATOM 1398 OG1 THR A 411 31.761 −6.416 −8.930 1.00 37.50 O ANISOU 1398 OG1 THR A 411 4710 4871 4664 84 −11 125 O ATOM 1399 CG2 THR A 411 33.018 −7.757 −7.351 1.00 36.73 C ANISOU 1399 CG2 THR A 411 4767 4595 4593 3 22 161 C ATOM 1400 C THR A 411 30.540 −8.758 −6.171 1.00 38.50 C ANISOU 1400 C THR A 411 4928 4919 4780 58 −33 132 C ATOM 1401 O THR A 411 30.377 −8.166 −5.098 1.00 38.70 O ANISOU 1401 O THR A 411 4944 5052 4708 98 23 156 O ATOM 1402 N VAL A 412 30.733 −10.070 −6.231 1.00 40.03 N ANISOU 1402 N VAL A 412 5107 5068 5034 48 −15 105 N ATOM 1403 CA VAL A 412 30.898 −10.879 −4.999 1.00 41.18 C ANISOU 1403 CA VAL A 412 5270 5215 5159 42 −14 107 C ATOM 1404 CB VAL A 412 29.671 −11.783 −4.715 1.00 40.91 C ANISOU 1404 CB VAL A 412 5223 5145 5175 45 −26 100 C ATOM 1405 CG1 VAL A 412 28.459 −10.957 −4.333 1.00 41.72 C ANISOU 1405 CG1 VAL A 412 5340 5253 5258 8 5 95 C ATOM 1406 CG2 VAL A 412 29.378 −12.688 −5.898 1.00 41.30 C ANISOU 1406 CG2 VAL A 412 5288 5262 5143 56 −93 79 C ATOM 1407 C VAL A 412 32.130 −11.777 −5.058 1.00 41.92 C ANISOU 1407 C VAL A 412 5342 5298 5284 55 2 92 C ATOM 1408 O VAL A 412 32.501 −12.230 −6.148 1.00 42.01 O ANISOU 1408 O VAL A 412 5411 5272 5277 51 −15 74 O ATOM 1409 N ASP A 413 32.741 −12.050 −3.895 1.00 43.17 N ANISOU 1409 N ASP A 413 5497 5486 5417 25 −27 44 N ATOM 1410 CA ASP A 413 33.828 −13.058 −3.794 1.00 44.45 C ANISOU 1410 CA ASP A 413 5685 5613 5589 20 −20 37 C ATOM 1411 CB ASP A 413 34.281 −13.268 −2.345 1.00 45.14 C ANISOU 1411 CB ASP A 413 5771 5740 5638 27 −24 76 C ATOM 1412 CG ASP A 413 34.976 −12.041 −1.745 1.00 46.83 C ANISOU 1412 CG ASP A 413 6015 5966 5809 −56 −80 52 C ATOM 1413 OD1 ASP A 413 35.210 −11.035 −2.457 1.00 48.90 O ANISOU 1413 OD1 ASP A 413 6156 6319 6102 −40 23 125 O ATOM 1414 OD2 ASP A 413 35.288 −12.084 −0.535 1.00 49.87 O ANISOU 1414 OD2 ASP A 413 6489 6388 6071 −31 −24 57 O ATOM 1415 C ASP A 413 33.345 −14.385 −4.372 1.00 44.46 C ANISOU 1415 C ASP A 413 5701 5572 5620 −1 2 46 C ATOM 1416 O ASP A 413 32.224 −14.799 −4.093 1.00 44.32 O ANISOU 1416 O ASP A 413 5691 5550 5596 4 41 42 O ATOM 1417 N LYS A 414 34.185 −15.020 −5.195 1.00 44.94 N ANISOU 1417 N LYS A 414 5770 5623 5682 8 −7 53 N ATOM 1418 CA LYS A 414 33.820 −16.224 −5.971 1.00 45.58 C ANISOU 1418 CA LYS A 414 5844 5751 5721 −21 −31 35 C ATOM 1419 CB LYS A 414 34.997 −16.633 −6.850 1.00 45.72 C ANISOU 1419 CB LYS A 414 5836 5796 5740 −19 −14 37 C ATOM 1420 CG LYS A 414 34.725 −17.758 −7.819 1.00 45.82 C ANISOU 1420 CG LYS A 414 5880 5705 5824 30 −23 30 C ATOM 1421 CD LYS A 414 35.966 −18.024 −8.634 1.00 47.84 C ANISOU 1421 CD LYS A 414 6077 6009 6089 18 52 −1 C ATOM 1422 CE LYS A 414 35.693 −18.974 −9.784 1.00 48.47 C ANISOU 1422 CE LYS A 414 6126 6229 6062 23 11 −100 C ATOM 1423 NZ LYS A 414 36.972 −19.359 −10.462 1.00 49.66 N ANISOU 1423 NZ LYS A 414 6253 6358 6256 15 38 −89 N ATOM 1424 C LYS A 414 33.401 −17.438 −5.138 1.00 46.06 C ANISOU 1424 C LYS A 414 5882 5808 5809 −53 −60 44 C ATOM 1425 O LYS A 414 32.556 −18.234 −5.566 1.00 47.07 O ANISOU 1425 O LYS A 414 5999 5982 5903 −24 −123 54 O ATOM 1426 N SER A 415 34.012 −17.606 −3.971 1.00 46.26 N ANISOU 1426 N SER A 415 5896 5861 5817 −68 −56 60 N ATOM 1427 CA SER A 415 33.679 −18.728 −3.088 1.00 46.61 C ANISOU 1427 CA SER A 415 5930 5891 5888 −68 −54 52 C ATOM 1428 CB SER A 415 34.552 −18.712 −1.810 1.00 46.81 C ANISOU 1428 CB SER A 415 5974 5905 5906 −59 −48 70 C ATOM 1429 OG SER A 415 34.803 −17.392 −1.321 1.00 46.84 O ANISOU 1429 OG SER A 415 6000 5875 5923 −99 −111 22 O ATOM 1430 C SER A 415 32.167 −18.732 −2.781 1.00 46.70 C ANISOU 1430 C SER A 415 5922 5886 5935 −46 −18 49 C ATOM 1431 O SER A 415 31.521 −19.780 −2.836 1.00 47.24 O ANISOU 1431 O SER A 415 5992 5952 6003 −61 −44 28 O ATOM 1432 N ARG A 416 31.617 −17.544 −2.512 1.00 46.83 N ANISOU 1432 N ARG A 416 5915 5904 5972 −34 −6 62 N ATOM 1433 CA ARG A 416 30.171 −17.325 −2.328 1.00 46.36 C ANISOU 1433 CA ARG A 416 5861 5859 5894 −34 0 66 C ATOM 1434 CB ARG A 416 29.887 −15.849 −2.088 1.00 46.60 C ANISOU 1434 CB ARG A 416 5918 5875 5912 0 41 69 C ATOM 1435 CG ARG A 416 29.867 −15.452 −0.644 1.00 47.76 C ANISOU 1435 CG ARG A 416 6143 6052 5950 −60 −4 53 C ATOM 1436 CD ARG A 416 29.723 −13.956 −0.492 1.00 47.81 C ANISOU 1436 CD ARG A 416 5941 6058 6165 −177 −24 4 C ATOM 1437 NE ARG A 416 30.998 −13.332 −0.127 1.00 51.98 N ANISOU 1437 NE ARG A 416 6596 6580 6574 64 83 221 N ATOM 1438 CZ ARG A 416 31.182 −12.562 0.945 1.00 48.85 C ANISOU 1438 CZ ARG A 416 5973 6438 6150 −125 66 −142 C ATOM 1439 NH1 ARG A 416 30.168 −12.312 1.763 1.00 53.54 N ANISOU 1439 NH1 ARG A 416 6843 6710 6787 7 1 33 N ATOM 1440 NH2 ARG A 416 32.379 −12.028 1.196 1.00 52.19 N ANISOU 1440 NH2 ARG A 416 6821 6562 6445 124 29 86 N ATOM 1441 C ARG A 416 29.326 −17.787 −3.503 1.00 46.10 C ANISOU 1441 C ARG A 416 5811 5841 5864 −26 −12 80 C ATOM 1442 O ARG A 416 28.229 −18.318 −3.299 1.00 46.58 O ANISOU 1442 O ARG A 416 5912 5852 5931 3 −22 129 O ATOM 1443 N TRP A 417 29.814 −17.566 −4.727 1.00 45.76 N ANISOU 1443 N TRP A 417 5753 5790 5841 −39 −6 47 N ATOM 1444 CA TRP A 417 29.109 −18.028 −5.933 1.00 45.44 C ANISOU 1444 CA TRP A 417 5708 5726 5828 −4 −12 32 C ATOM 1445 CB TRP A 417 29.750 −17.485 −7.222 1.00 43.60 C ANISOU 1445 CB TRP A 417 5388 5589 5589 11 −113 20 C ATOM 1446 CG TRP A 417 29.056 −17.925 −8.508 1.00 43.73 C ANISOU 1446 CG TRP A 417 5357 5558 5700 39 −41 38 C ATOM 1447 CD1 TRP A 417 29.598 −18.674 −9.537 1.00 41.28 C ANISOU 1447 CD1 TRP A 417 4777 5490 5416 116 −99 22 C ATOM 1448 NE1 TRP A 417 28.662 −18.887 −10.523 1.00 44.32 N ANISOU 1448 NE1 TRP A 417 5725 5455 5657 −184 23 161 N ATOM 1449 CE2 TRP A 417 27.488 −18.279 −10.152 1.00 41.66 C ANISOU 1449 CE2 TRP A 417 5146 5311 5372 46 −8 −11 C ATOM 1450 CD2 TRP A 417 27.698 −17.665 −8.888 1.00 43.53 C ANISOU 1450 CD2 TRP A 417 5499 5455 5584 22 36 94 C ATOM 1451 CE3 TRP A 417 26.634 −16.972 −8.286 1.00 42.96 C ANISOU 1451 CE3 TRP A 417 5359 5442 5518 17 14 67 C ATOM 1452 CZ3 TRP A 417 25.417 −16.905 −8.962 1.00 42.84 C ANISOU 1452 CZ3 TRP A 417 5360 5442 5473 −41 61 −2 C ATOM 1453 CH2 TRP A 417 25.246 −17.517 −10.217 1.00 43.50 C ANISOU 1453 CH2 TRP A 417 5561 5399 5567 −97 121 76 C ATOM 1454 CZ2 TRP A 417 26.263 −18.206 −10.824 1.00 42.87 C ANISOU 1454 CZ2 TRP A 417 5358 5337 5590 −6 99 73 C ATOM 1455 C TRP A 417 29.072 −19.558 −5.927 1.00 46.18 C ANISOU 1455 C TRP A 417 5795 5803 5946 7 −35 30 C ATOM 1456 O TRP A 417 28.009 −20.167 −6.166 1.00 46.90 O ANISOU 1456 O TRP A 417 5817 5939 6064 −1 −68 67 O ATOM 1457 N GLN A 418 30.217 −20.167 −5.598 1.00 46.23 N ANISOU 1457 N GLN A 418 5783 5839 5941 48 −14 42 N ATOM 1458 CA GLN A 418 30.356 −21.636 −5.575 1.00 46.42 C ANISOU 1458 CA GLN A 418 5870 5841 5926 17 −2 41 C ATOM 1459 CB GLN A 418 31.842 −22.049 −5.700 1.00 46.09 C ANISOU 1459 CB GLN A 418 5815 5825 5872 36 19 53 C ATOM 1460 CG GLN A 418 32.458 −21.757 −7.083 1.00 46.48 C ANISOU 1460 CG GLN A 418 5847 5833 5976 34 73 −10 C ATOM 1461 CD GLN A 418 34.002 −21.715 −7.095 1.00 46.67 C ANISOU 1461 CD GLN A 418 5898 5914 5920 16 24 20 C ATOM 1462 OE1 GLN A 418 34.665 −21.686 −6.048 1.00 47.82 O ANISOU 1462 OE1 GLN A 418 6033 6136 6000 53 −33 66 O ATOM 1463 NE2 GLN A 418 34.571 −21.701 −8.297 1.00 47.35 N ANISOU 1463 NE2 GLN A 418 5961 6023 6004 33 66 26 N ATOM 1464 C GLN A 418 29.653 −22.299 −4.363 1.00 46.78 C ANISOU 1464 C GLN A 418 5905 5866 6002 −9 −3 20 C ATOM 1465 O GLN A 418 29.001 −23.337 −4.506 1.00 47.00 O ANISOU 1465 O GLN A 418 5932 5847 6078 −40 −29 35 O ATOM 1466 N GLN A 419 29.777 −21.696 −3.180 1.00 47.14 N ANISOU 1466 N GLN A 419 6009 5940 5961 −18 −9 36 N ATOM 1467 CA GLN A 419 29.014 −22.148 −2.017 1.00 48.11 C ANISOU 1467 CA GLN A 419 6130 6033 6117 −16 −35 52 C ATOM 1468 CB GLN A 419 29.192 −21.182 −0.827 1.00 47.74 C ANISOU 1468 CB GLN A 419 6122 5956 6058 −21 −65 7 C ATOM 1469 CG GLN A 419 30.584 −21.313 −0.189 1.00 48.88 C ANISOU 1469 CG GLN A 419 6202 6136 6231 −31 −83 18 C ATOM 1470 CD GLN A 419 30.835 −20.383 1.010 1.00 48.22 C ANISOU 1470 CD GLN A 419 6253 6005 6062 61 −160 −28 C ATOM 1471 OE1 GLN A 419 30.374 −19.232 1.050 1.00 51.84 O ANISOU 1471 OE1 GLN A 419 6788 6288 6621 72 −49 −14 O ATOM 1472 NE2 GLN A 419 31.595 −20.883 1.981 1.00 48.48 N ANISOU 1472 NE2 GLN A 419 6320 6025 6074 22 −59 −61 N ATOM 1473 C GLN A 419 27.538 −22.358 −2.393 1.00 47.88 C ANISOU 1473 C GLN A 419 6103 5976 6111 −20 −69 81 C ATOM 1474 O GLN A 419 26.977 −23.449 −2.213 1.00 48.69 O ANISOU 1474 O GLN A 419 6259 5993 6247 −19 −60 110 O ATOM 1475 N GLY A 420 26.911 −21.333 −2.954 1.00 47.48 N ANISOU 1475 N GLY A 420 6057 5915 6069 −12 −64 79 N ATOM 1476 CA GLY A 420 25.605 −21.536 −3.553 1.00 46.95 C ANISOU 1476 CA GLY A 420 5936 5893 6010 15 −32 74 C ATOM 1477 C GLY A 420 24.588 −20.527 −3.120 1.00 46.55 C ANISOU 1477 C GLY A 420 5880 5886 5920 12 −26 49 C ATOM 1478 O GLY A 420 23.394 −20.701 −3.397 1.00 46.93 O ANISOU 1478 O GLY A 420 5889 5969 5972 −9 −8 74 O ATOM 1479 N ASN A 421 25.070 −19.488 −2.433 1.00 46.34 N ANISOU 1479 N ASN A 421 5839 5872 5896 40 −50 46 N ATOM 1480 CA ASN A 421 24.289 −18.301 −2.077 1.00 45.85 C ANISOU 1480 CA ASN A 421 5786 5865 5770 64 −39 9 C ATOM 1481 CB ASN A 421 25.211 −17.091 −1.903 1.00 45.59 C ANISOU 1481 CB ASN A 421 5783 5824 5712 51 −56 13 C ATOM 1482 CG ASN A 421 25.560 −16.808 −0.438 1.00 46.44 C ANISOU 1482 CG ASN A 421 5950 5950 5741 86 −2 −15 C ATOM 1483 OD1 ASN A 421 26.110 −17.659 0.284 1.00 43.48 O ANISOU 1483 OD1 ASN A 421 5884 5482 5152 142 48 64 O ATOM 1484 ND2 ASN A 421 25.277 −15.577 −0.009 1.00 47.25 N ANISOU 1484 ND2 ASN A 421 6145 5952 5855 165 −93 −103 N ATOM 1485 C ASN A 421 23.213 −17.924 −3.095 1.00 45.79 C ANISOU 1485 C ASN A 421 5772 5862 5764 76 −20 43 C ATOM 1486 O ASN A 421 23.401 −18.062 −4.327 1.00 45.60 O ANISOU 1486 O ASN A 421 5768 5864 5693 63 −16 71 O ATOM 1487 N VAL A 422 22.089 −17.454 −2.553 1.00 45.55 N ANISOU 1487 N VAL A 422 5739 5837 5731 49 −11 67 N ATOM 1488 CA VAL A 422 21.035 −16.834 −3.337 1.00 45.24 C ANISOU 1488 CA VAL A 422 5704 5739 5744 26 10 70 C ATOM 1489 CB VAL A 422 19.661 −17.027 −2.690 1.00 45.54 C ANISOU 1489 CB VAL A 422 5700 5790 5810 24 9 65 C ATOM 1490 CG1 VAL A 422 18.638 −16.039 −3.274 1.00 46.04 C ANISOU 1490 CG1 VAL A 422 5848 5757 5887 73 42 96 C ATOM 1491 CG2 VAL A 422 19.197 −18.476 −2.879 1.00 45.88 C ANISOU 1491 CG2 VAL A 422 5756 5710 5965 34 −24 7 C ATOM 1492 C VAL A 422 21.346 −15.357 −3.459 1.00 44.78 C ANISOU 1492 C VAL A 422 5682 5693 5638 10 15 94 C ATOM 1493 O VAL A 422 21.613 −14.670 −2.457 1.00 44.88 O ANISOU 1493 O VAL A 422 5745 5738 5570 0 98 124 O ATOM 1494 N PHE A 423 21.369 −14.880 −4.698 1.00 44.38 N ANISOU 1494 N PHE A 423 5592 5648 5622 7 −27 107 N ATOM 1495 CA PHE A 423 21.495 −13.452 −4.933 1.00 43.56 C ANISOU 1495 CA PHE A 423 5497 5571 5482 0 −24 111 C ATOM 1496 CB PHE A 423 22.798 −13.133 −5.646 1.00 43.29 C ANISOU 1496 CB PHE A 423 5394 5556 5497 −7 −79 107 C ATOM 1497 CG PHE A 423 24.008 −13.224 −4.759 1.00 44.33 C ANISOU 1497 CG PHE A 423 5633 5632 5577 −92 29 161 C ATOM 1498 CD1 PHE A 423 24.936 −14.249 −4.937 1.00 42.42 C ANISOU 1498 CD1 PHE A 423 5139 5468 5509 38 −37 193 C ATOM 1499 CE1 PHE A 423 26.047 −14.339 −4.131 1.00 42.33 C ANISOU 1499 CE1 PHE A 423 5468 5273 5342 −127 119 −44 C ATOM 1500 CZ PHE A 423 26.245 −13.401 −3.133 1.00 45.72 C ANISOU 1500 CZ PHE A 423 5685 6031 5653 −193 105 211 C ATOM 1501 CE2 PHE A 423 25.328 −12.362 −2.944 1.00 41.37 C ANISOU 1501 CE2 PHE A 423 4978 5384 5355 209 −92 166 C ATOM 1502 CD2 PHE A 423 24.217 −12.288 −3.751 1.00 43.86 C ANISOU 1502 CD2 PHE A 423 5670 5522 5470 −44 136 92 C ATOM 1503 C PHE A 423 20.288 −12.976 −5.723 1.00 43.24 C ANISOU 1503 C PHE A 423 5452 5530 5446 −1 −11 102 C ATOM 1504 O PHE A 423 19.889 −13.612 −6.714 1.00 42.35 O ANISOU 1504 O PHE A 423 5366 5482 5242 −37 −48 112 O ATOM 1505 N SER A 424 19.699 −11.880 −5.251 1.00 42.62 N ANISOU 1505 N SER A 424 5361 5473 5357 9 17 73 N ATOM 1506 CA SER A 424 18.502 −11.336 −5.875 1.00 42.62 C ANISOU 1506 CA SER A 424 5334 5509 5350 15 32 50 C ATOM 1507 CB SER A 424 17.269 −11.580 −4.983 1.00 42.80 C ANISOU 1507 CB SER A 424 5346 5561 5353 30 34 25 C ATOM 1508 OG SER A 424 17.321 −10.787 −3.812 1.00 42.53 O ANISOU 1508 OG SER A 424 5276 5654 5226 85 74 39 O ATOM 1509 C SER A 424 18.626 −9.859 −6.300 1.00 42.35 C ANISOU 1509 C SER A 424 5296 5467 5328 20 18 77 C ATOM 1510 O SER A 424 19.033 −8.992 −5.518 1.00 41.38 O ANISOU 1510 O SER A 424 5174 5364 5184 −26 −2 71 O ATOM 1511 N CYS A 425 18.282 −9.603 −7.563 1.00 42.32 N ANISOU 1511 N CYS A 425 5228 5490 5359 38 3 113 N ATOM 1512 CA CYS A 425 18.182 −8.238 −8.086 1.00 42.32 C ANISOU 1512 CA CYS A 425 5236 5477 5364 15 4 122 C ATOM 1513 CB CYS A 425 18.691 −8.193 −9.527 1.00 42.07 C ANISOU 1513 CB CYS A 425 5168 5425 5391 5 −28 131 C ATOM 1514 SG CYS A 425 18.168 −6.769 −10.529 1.00 41.39 S ANISOU 1514 SG CYS A 425 4930 5467 5327 77 −26 166 S ATOM 1515 C CYS A 425 16.729 −7.761 −7.995 1.00 42.83 C ANISOU 1515 C CYS A 425 5386 5497 5390 14 2 110 C ATOM 1516 O CYS A 425 15.813 −8.361 −8.585 1.00 42.19 O ANISOU 1516 O CYS A 425 5252 5457 5320 −2 48 124 O ATOM 1517 N SER A 426 16.521 −6.693 −7.231 1.00 43.33 N ANISOU 1517 N SER A 426 5489 5511 5460 6 −17 101 N ATOM 1518 CA SER A 426 15.185 −6.185 −7.025 1.00 43.91 C ANISOU 1518 CA SER A 426 5550 5598 5534 −21 −15 67 C ATOM 1519 CB SER A 426 14.815 −6.219 −5.538 1.00 44.36 C ANISOU 1519 CB SER A 426 5616 5660 5575 −47 −55 69 C ATOM 1520 OG SER A 426 15.437 −5.186 −4.808 1.00 45.92 O ANISOU 1520 OG SER A 426 5898 5798 5752 −91 −95 43 O ATOM 1521 C SER A 426 14.983 −4.801 −7.660 1.00 43.57 C ANISOU 1521 C SER A 426 5506 5553 5493 −13 −31 57 C ATOM 1522 O SER A 426 15.711 −3.850 −7.371 1.00 43.22 O ANISOU 1522 O SER A 426 5456 5506 5457 −9 −46 60 O ATOM 1523 N VAL A 427 13.976 −4.725 −8.528 1.00 42.97 N ANISOU 1523 N VAL A 427 5449 5498 5380 −15 −21 49 N ATOM 1524 CA VAL A 427 13.734 −3.579 −9.400 1.00 42.05 C ANISOU 1524 CA VAL A 427 5340 5430 5205 0 −15 33 C ATOM 1525 CB VAL A 427 13.627 −4.051 −10.884 1.00 41.86 C ANISOU 1525 CB VAL A 427 5301 5420 5181 17 −2 −11 C ATOM 1526 CG1 VAL A 427 13.382 −2.874 −11.821 1.00 40.21 C ANISOU 1526 CG1 VAL A 427 5056 5232 4987 −44 57 −23 C ATOM 1527 CG2 VAL A 427 14.872 −4.811 −11.296 1.00 41.53 C ANISOU 1527 CG2 VAL A 427 5292 5317 5170 0 −112 30 C ATOM 1528 C VAL A 427 12.453 −2.820 −9.041 1.00 41.50 C ANISOU 1528 C VAL A 427 5362 5341 5064 −13 8 28 C ATOM 1529 O VAL A 427 11.390 −3.408 −8.880 1.00 40.96 O ANISOU 1529 O VAL A 427 5279 5330 4951 −6 48 98 O ATOM 1530 N MET A 428 12.559 −1.500 −8.969 1.00 41.12 N ANISOU 1530 N MET A 428 5318 5327 4978 −22 7 9 N ATOM 1531 CA MET A 428 11.395 −0.647 −8.805 1.00 40.47 C ANISOU 1531 CA MET A 428 5291 5197 4889 −14 60 −23 C ATOM 1532 CB MET A 428 11.585 0.281 −7.620 1.00 40.36 C ANISOU 1532 CB MET A 428 5204 5222 4908 −56 23 −2 C ATOM 1533 CG MET A 428 12.037 −0.413 −6.373 1.00 41.31 C ANISOU 1533 CG MET A 428 5409 5218 5068 −29 52 −5 C ATOM 1534 SD MET A 428 11.727 0.663 −4.974 1.00 42.39 S ANISOU 1534 SD MET A 428 5767 5498 4839 −41 119 −25 S ATOM 1535 CE MET A 428 9.947 0.788 −5.114 1.00 40.75 C ANISOU 1535 CE MET A 428 5472 5091 4920 29 70 102 C ATOM 1536 C MET A 428 11.096 0.175 −10.057 1.00 39.15 C ANISOU 1536 C MET A 428 5085 5011 4777 14 40 −108 C ATOM 1537 O MET A 428 11.944 0.908 −10.555 1.00 38.37 O ANISOU 1537 O MET A 428 5039 4855 4683 13 60 −72 O ATOM 1538 N HIS A 429 9.876 0.038 −10.556 1.00 37.88 N ANISOU 1538 N HIS A 429 4935 4841 4613 56 57 −125 N ATOM 1539 CA HIS A 429 9.427 0.779 −11.730 1.00 36.90 C ANISOU 1539 CA HIS A 429 4763 4738 4517 31 43 −119 C ATOM 1540 CB HIS A 429 9.829 0.086 −13.033 1.00 35.73 C ANISOU 1540 CB HIS A 429 4590 4600 4386 −2 24 −67 C ATOM 1541 CG HIS A 429 9.560 0.914 −14.246 1.00 33.46 C ANISOU 1541 CG HIS A 429 4228 4253 4232 12 111 −213 C ATOM 1542 ND1 HIS A 429 8.334 0.950 −14.870 1.00 31.22 N ANISOU 1542 ND1 HIS A 429 4260 3907 3694 25 43 −229 N ATOM 1543 CE1 HIS A 429 8.385 1.778 −15.894 1.00 30.83 C ANISOU 1543 CE1 HIS A 429 4069 3810 3833 −36 51 −163 C ATOM 1544 NE2 HIS A 429 9.602 2.278 −15.960 1.00 31.02 N ANISOU 1544 NE2 HIS A 429 4048 3977 3758 10 −105 −163 N ATOM 1545 CD2 HIS A 429 10.353 1.763 −14.935 1.00 32.35 C ANISOU 1545 CD2 HIS A 429 4277 3969 4045 86 47 −124 C ATOM 1546 C HIS A 429 7.929 0.843 −11.664 1.00 36.82 C ANISOU 1546 C HIS A 429 4799 4771 4418 26 9 −122 C ATOM 1547 O HIS A 429 7.297 −0.115 −11.229 1.00 36.28 O ANISOU 1547 O HIS A 429 4722 4796 4265 10 −12 −179 O ATOM 1548 N GLU A 430 7.374 1.964 −12.119 1.00 36.71 N ANISOU 1548 N GLU A 430 4815 4761 4371 55 19 −106 N ATOM 1549 CA GLU A 430 5.934 2.203 −12.098 1.00 36.80 C ANISOU 1549 CA GLU A 430 4811 4731 4437 41 6 −42 C ATOM 1550 CB GLU A 430 5.594 3.547 −12.773 1.00 36.76 C ANISOU 1550 CB GLU A 430 4791 4744 4430 −9 −30 −31 C ATOM 1551 CG GLU A 430 5.694 3.553 −14.307 1.00 35.19 C ANISOU 1551 CG GLU A 430 4490 4486 4392 25 72 100 C ATOM 1552 CD GLU A 430 5.001 4.742 −14.895 1.00 34.83 C ANISOU 1552 CD GLU A 430 4558 4546 4128 −71 229 48 C ATOM 1553 OE1 GLU A 430 5.453 5.862 −14.630 1.00 30.64 O ANISOU 1553 OE1 GLU A 430 4122 4033 3484 −100 187 82 O ATOM 1554 OE2 GLU A 430 3.991 4.560 −15.606 1.00 34.10 O ANISOU 1554 OE2 GLU A 430 4321 4585 4048 −96 202 −27 O ATOM 1555 C GLU A 430 5.110 1.099 −12.716 1.00 36.55 C ANISOU 1555 C GLU A 430 4793 4631 4463 48 −12 −17 C ATOM 1556 O GLU A 430 4.035 0.794 −12.223 1.00 36.45 O ANISOU 1556 O GLU A 430 4876 4636 4334 94 23 −111 O ATOM 1557 N ALA A 431 5.613 0.513 −13.803 1.00 36.85 N ANISOU 1557 N ALA A 431 4769 4635 4595 51 −26 26 N ATOM 1558 CA ALA A 431 4.836 −0.427 −14.622 1.00 36.97 C ANISOU 1558 CA ALA A 431 4763 4665 4617 35 0 −11 C ATOM 1559 CB ALA A 431 5.256 −0.312 −16.072 1.00 36.63 C ANISOU 1559 CB ALA A 431 4680 4637 4600 53 −51 −6 C ATOM 1560 C ALA A 431 4.901 −1.891 −14.129 1.00 37.11 C ANISOU 1560 C ALA A 431 4752 4712 4633 −16 12 −11 C ATOM 1561 O ALA A 431 4.366 −2.798 −14.764 1.00 37.32 O ANISOU 1561 O ALA A 431 4770 4750 4656 −49 59 −15 O ATOM 1562 N LEU A 432 5.562 −2.117 −13.003 1.00 36.79 N ANISOU 1562 N LEU A 432 4741 4648 4588 −2 5 7 N ATOM 1563 CA LEU A 432 5.501 −3.419 −12.333 1.00 37.15 C ANISOU 1563 CA LEU A 432 4763 4747 4605 −3 −9 8 C ATOM 1564 CB LEU A 432 6.813 −3.728 −11.587 1.00 36.16 C ANISOU 1564 CB LEU A 432 4697 4593 4448 14 −19 45 C ATOM 1565 CG LEU A 432 8.039 −3.920 −12.494 1.00 35.48 C ANISOU 1565 CG LEU A 432 4565 4483 4433 11 −70 72 C ATOM 1566 CD1 LEU A 432 9.375 −3.695 −11.782 1.00 33.76 C ANISOU 1566 CD1 LEU A 432 4494 4326 4005 44 −80 116 C ATOM 1567 CD2 LEU A 432 8.019 −5.273 −13.221 1.00 34.94 C ANISOU 1567 CD2 LEU A 432 4448 4511 4315 30 −64 152 C ATOM 1568 C LEU A 432 4.287 −3.470 −11.401 1.00 37.38 C ANISOU 1568 C LEU A 432 4819 4804 4578 −12 −9 34 C ATOM 1569 O LEU A 432 3.802 −2.445 −10.931 1.00 37.28 O ANISOU 1569 O LEU A 432 4766 4889 4509 32 1 67 O ATOM 1570 N HIS A 433 3.768 −4.663 −11.178 1.00 38.17 N ANISOU 1570 N HIS A 433 4911 4926 4663 −24 −11 39 N ATOM 1571 CA HIS A 433 2.747 −4.869 −10.161 1.00 38.86 C ANISOU 1571 CA HIS A 433 4958 5015 4789 −27 −2 49 C ATOM 1572 CB HIS A 433 2.447 −6.358 −10.078 1.00 39.24 C ANISOU 1572 CB HIS A 433 4992 5068 4847 −27 6 41 C ATOM 1573 CG HIS A 433 1.480 −6.722 −9.003 1.00 41.29 C ANISOU 1573 CG HIS A 433 5182 5325 5179 −26 27 73 C ATOM 1574 ND1 HIS A 433 0.117 −6.574 −9.150 1.00 42.44 N ANISOU 1574 ND1 HIS A 433 5238 5453 5433 −68 34 41 N ATOM 1575 CE1 HIS A 433 −0.482 −6.986 −8.046 1.00 42.23 C ANISOU 1575 CE1 HIS A 433 5381 5485 5176 10 −17 −10 C ATOM 1576 NE2 HIS A 433 0.443 −7.399 −7.197 1.00 41.94 N ANISOU 1576 NE2 HIS A 433 5411 5442 5079 −19 12 −1 N ATOM 1577 CD2 HIS A 433 1.678 −7.241 −7.769 1.00 40.89 C ANISOU 1577 CD2 HIS A 433 5136 5376 5025 −8 −29 20 C ATOM 1578 C HIS A 433 3.279 −4.308 −8.824 1.00 38.60 C ANISOU 1578 C HIS A 433 4959 4969 4737 −33 8 95 C ATOM 1579 O HIS A 433 4.392 −4.632 −8.416 1.00 38.55 O ANISOU 1579 O HIS A 433 4974 5004 4668 −49 −28 116 O ATOM 1580 N ASN A 434 2.522 −3.420 −8.187 1.00 38.26 N ANISOU 1580 N ASN A 434 4920 4949 4665 −74 17 108 N ATOM 1581 CA ASN A 434 2.994 −2.709 −6.994 1.00 38.50 C ANISOU 1581 CA ASN A 434 4948 4950 4727 −75 13 90 C ATOM 1582 CB ASN A 434 2.999 −3.639 −5.763 1.00 38.77 C ANISOU 1582 CB ASN A 434 4941 5054 4733 −120 20 41 C ATOM 1583 CG ASN A 434 1.631 −4.134 −5.413 1.00 38.47 C ANISOU 1583 CG ASN A 434 4913 5088 4614 −105 23 7 C ATOM 1584 OD1 ASN A 434 1.466 −5.285 −5.048 1.00 38.40 O ANISOU 1584 OD1 ASN A 434 5064 4785 4740 −130 −43 51 O ATOM 1585 ND2 ASN A 434 0.635 −3.275 −5.548 1.00 35.57 N ANISOU 1585 ND2 ASN A 434 4416 4664 4435 −105 161 −54 N ATOM 1586 C ASN A 434 4.372 −2.064 −7.143 1.00 38.65 C ANISOU 1586 C ASN A 434 4962 4997 4727 −89 12 73 C ATOM 1587 O ASN A 434 5.092 −1.904 −6.156 1.00 38.37 O ANISOU 1587 O ASN A 434 4948 4953 4677 −158 44 68 O ATOM 1588 N HIS A 435 4.729 −1.721 −8.384 1.00 38.28 N ANISOU 1588 N HIS A 435 4930 4968 4646 −59 −13 18 N ATOM 1589 CA HIS A 435 5.963 −1.016 −8.726 1.00 37.58 C ANISOU 1589 CA HIS A 435 4881 4891 4504 −25 5 35 C ATOM 1590 CB HIS A 435 5.952 0.417 −8.155 1.00 37.74 C ANISOU 1590 CB HIS A 435 4898 4903 4537 −23 −29 −1 C ATOM 1591 CG HIS A 435 4.584 1.032 −8.082 1.00 37.92 C ANISOU 1591 CG HIS A 435 4873 4934 4598 −13 14 −80 C ATOM 1592 ND1 HIS A 435 3.749 1.135 −9.174 1.00 37.42 N ANISOU 1592 ND1 HIS A 435 4902 4856 4459 161 35 −122 N ATOM 1593 CE1 HIS A 435 2.607 1.686 −8.810 1.00 38.63 C ANISOU 1593 CE1 HIS A 435 4913 4743 5020 62 15 82 C ATOM 1594 NE2 HIS A 435 2.681 1.979 −7.524 1.00 36.99 N ANISOU 1594 NE2 HIS A 435 4821 4801 4432 32 −57 3 N ATOM 1595 CD2 HIS A 435 3.903 1.573 −7.044 1.00 38.62 C ANISOU 1595 CD2 HIS A 435 4975 4958 4739 −34 6 −67 C ATOM 1596 C HIS A 435 7.236 −1.770 −8.330 1.00 37.17 C ANISOU 1596 C HIS A 435 4870 4880 4372 −25 31 22 C ATOM 1597 O HIS A 435 8.273 −1.176 −8.127 1.00 35.86 O ANISOU 1597 O HIS A 435 4733 4829 4063 47 22 64 O ATOM 1598 N TYR A 436 7.153 −3.088 −8.248 1.00 37.91 N ANISOU 1598 N TYR A 436 4963 4964 4476 1 14 15 N ATOM 1599 CA TYR A 436 8.235 −3.873 −7.684 1.00 38.88 C ANISOU 1599 CA TYR A 436 5029 4992 4751 2 12 −9 C ATOM 1600 CB TYR A 436 8.058 −3.997 −6.158 1.00 38.93 C ANISOU 1600 CB TYR A 436 5047 4980 4765 8 −25 −6 C ATOM 1601 CG TYR A 436 9.235 −4.641 −5.455 1.00 39.72 C ANISOU 1601 CG TYR A 436 5116 5014 4959 60 6 −69 C ATOM 1602 CD1 TYR A 436 9.202 −5.986 −5.059 1.00 40.15 C ANISOU 1602 CD1 TYR A 436 5192 5178 4884 57 49 35 C ATOM 1603 CE1 TYR A 436 10.302 −6.573 −4.438 1.00 39.78 C ANISOU 1603 CE1 TYR A 436 5150 5156 4806 17 −21 78 C ATOM 1604 CZ TYR A 436 11.426 −5.797 −4.193 1.00 40.05 C ANISOU 1604 CZ TYR A 436 5151 5177 4887 17 10 47 C ATOM 1605 OH TYR A 436 12.530 −6.318 −3.580 1.00 39.84 O ANISOU 1605 OH TYR A 436 5240 5038 4856 63 −12 9 O ATOM 1606 CE2 TYR A 436 11.465 −4.467 −4.566 1.00 40.35 C ANISOU 1606 CE2 TYR A 436 5160 5135 5036 62 −59 13 C ATOM 1607 CD2 TYR A 436 10.389 −3.905 −5.195 1.00 40.97 C ANISOU 1607 CD2 TYR A 436 5145 5122 5300 80 3 −58 C ATOM 1608 C TYR A 436 8.303 −5.264 −8.287 1.00 39.02 C ANISOU 1608 C TYR A 436 5083 5005 4736 −49 −25 −2 C ATOM 1609 O TYR A 436 7.289 −5.871 −8.531 1.00 38.66 O ANISOU 1609 O TYR A 436 5121 4885 4682 −64 −60 4 O ATOM 1610 N THR A 437 9.514 −5.755 −8.503 1.00 39.50 N ANISOU 1610 N THR A 437 5159 5021 4828 −10 2 −25 N ATOM 1611 CA THR A 437 9.734 −7.163 −8.753 1.00 40.83 C ANISOU 1611 CA THR A 437 5264 5167 5080 −30 15 33 C ATOM 1612 CB THR A 437 9.495 −7.556 −10.250 1.00 40.93 C ANISOU 1612 CB THR A 437 5246 5166 5137 −33 3 −17 C ATOM 1613 OG1 THR A 437 9.270 −8.968 −10.336 1.00 41.06 O ANISOU 1613 OG1 THR A 437 5236 5079 5286 15 103 −13 O ATOM 1614 CG2 THR A 437 10.665 −7.153 −11.145 1.00 39.50 C ANISOU 1614 CG2 THR A 437 5153 4957 4898 −9 5 −1 C ATOM 1615 C THR A 437 11.121 −7.579 −8.248 1.00 41.64 C ANISOU 1615 C THR A 437 5343 5262 5214 −19 −19 68 C ATOM 1616 O THR A 437 11.926 −6.731 −7.857 1.00 41.69 O ANISOU 1616 O THR A 437 5358 5239 5240 −28 −17 144 O ATOM 1617 N GLN A 438 11.371 −8.886 −8.250 1.00 42.72 N ANISOU 1617 N GLN A 438 5471 5368 5392 −15 17 61 N ATOM 1618 CA GLN A 438 12.573 −9.499 −7.668 1.00 43.76 C ANISOU 1618 CA GLN A 438 5564 5521 5541 0 12 72 C ATOM 1619 CB GLN A 438 12.260 −10.041 −6.266 1.00 43.80 C ANISOU 1619 CB GLN A 438 5570 5515 5557 21 −19 59 C ATOM 1620 CG GLN A 438 13.471 −10.272 −5.363 1.00 45.09 C ANISOU 1620 CG GLN A 438 5780 5691 5659 13 21 68 C ATOM 1621 CD GLN A 438 13.088 −10.493 −3.894 1.00 44.91 C ANISOU 1621 CD GLN A 438 5851 5578 5634 4 82 72 C ATOM 1622 OE1 GLN A 438 12.943 −11.640 −3.453 1.00 46.59 O ANISOU 1622 OE1 GLN A 438 5980 5851 5871 −13 157 97 O ATOM 1623 NE2 GLN A 438 12.927 −9.394 −3.132 1.00 45.98 N ANISOU 1623 NE2 GLN A 438 5903 5784 5783 −47 135 46 N ATOM 1624 C GLN A 438 12.951 −10.641 −8.584 1.00 43.72 C ANISOU 1624 C GLN A 438 5579 5440 5593 3 −9 94 C ATOM 1625 O GLN A 438 12.083 −11.348 −9.079 1.00 43.70 O ANISOU 1625 O GLN A 438 5600 5396 5605 −1 −19 100 O ATOM 1626 N LYS A 439 14.239 −10.788 −8.859 1.00 44.68 N ANISOU 1626 N LYS A 439 5693 5568 5715 −2 −26 96 N ATOM 1627 CA LYS A 439 14.741 −11.933 −9.615 1.00 45.43 C ANISOU 1627 CA LYS A 439 5800 5662 5798 −16 −9 70 C ATOM 1628 CB LYS A 439 15.058 −11.555 −11.064 1.00 45.89 C ANISOU 1628 CB LYS A 439 5825 5736 5873 −5 −9 40 C ATOM 1629 CG LYS A 439 13.856 −11.073 −11.880 1.00 46.21 C ANISOU 1629 CG LYS A 439 5900 5781 5874 51 −46 49 C ATOM 1630 CD LYS A 439 12.907 −12.194 −12.253 1.00 46.24 C ANISOU 1630 CD LYS A 439 5775 5776 6016 −18 −95 97 C ATOM 1631 CE LYS A 439 11.616 −11.657 −12.829 1.00 47.33 C ANISOU 1631 CE LYS A 439 6039 5979 5962 −62 19 23 C ATOM 1632 NZ LYS A 439 10.782 −12.775 −13.332 1.00 47.50 N ANISOU 1632 NZ LYS A 439 6069 6016 5962 −55 −46 41 N ATOM 1633 C LYS A 439 15.969 −12.490 −8.917 1.00 46.09 C ANISOU 1633 C LYS A 439 5848 5746 5916 −7 −7 78 C ATOM 1634 O LYS A 439 16.907 −11.749 −8.596 1.00 46.55 O ANISOU 1634 O LYS A 439 5959 5751 5975 6 −6 117 O ATOM 1635 N SER A 440 15.946 −13.796 −8.655 1.00 46.72 N ANISOU 1635 N SER A 440 5934 5839 5979 −31 −11 81 N ATOM 1636 CA SER A 440 17.032 −14.454 −7.942 1.00 46.85 C ANISOU 1636 CA SER A 440 5928 5897 5975 −7 −17 88 C ATOM 1637 CB SER A 440 16.496 −15.469 −6.913 1.00 47.28 C ANISOU 1637 CB SER A 440 5941 6005 6016 −9 −20 46 C ATOM 1638 OG SER A 440 16.183 −14.836 −5.671 1.00 48.78 O ANISOU 1638 OG SER A 440 6002 6424 6105 −24 17 60 O ATOM 1639 C SER A 440 17.973 −15.124 −8.918 1.00 46.89 C ANISOU 1639 C SER A 440 5980 5868 5969 −17 −17 95 C ATOM 1640 O SER A 440 17.545 −15.633 −9.959 1.00 47.05 O ANISOU 1640 O SER A 440 6023 5846 6007 −34 −57 109 O ATOM 1641 N LEU A 441 19.258 −15.097 −8.571 1.00 47.27 N ANISOU 1641 N LEU A 441 6010 5908 6042 16 −21 93 N ATOM 1642 CA LEU A 441 20.323 −15.741 −9.329 1.00 47.61 C ANISOU 1642 CA LEU A 441 6027 5975 6087 14 1 78 C ATOM 1643 CB LEU A 441 21.264 −14.677 −9.907 1.00 47.64 C ANISOU 1643 CB LEU A 441 6073 5959 6066 37 7 102 C ATOM 1644 CG LEU A 441 22.391 −15.101 −10.862 1.00 47.04 C ANISOU 1644 CG LEU A 441 5967 5906 5997 40 6 66 C ATOM 1645 CD1 LEU A 441 21.849 −15.691 −12.147 1.00 47.09 C ANISOU 1645 CD1 LEU A 441 6088 5875 5927 76 24 60 C ATOM 1646 CD2 LEU A 441 23.266 −13.921 −11.162 1.00 47.11 C ANISOU 1646 CD2 LEU A 441 5985 5891 6020 25 −6 88 C ATOM 1647 C LEU A 441 21.101 −16.740 −8.437 1.00 48.31 C ANISOU 1647 C LEU A 441 6136 6022 6196 33 −7 89 C ATOM 1648 O LEU A 441 21.434 −16.440 −7.269 1.00 47.92 O ANISOU 1648 O LEU A 441 6026 5982 6196 0 −72 71 O ATOM 1649 N SER A 442 21.366 −17.924 −8.998 1.00 49.31 N ANISOU 1649 N SER A 442 6259 6162 6314 38 26 67 N ATOM 1650 CA SER A 442 22.025 −19.032 −8.263 1.00 50.46 C ANISOU 1650 CA SER A 442 6432 6297 6442 43 18 55 C ATOM 1651 CB SER A 442 20.983 −19.965 −7.635 1.00 50.19 C ANISOU 1651 CB SER A 442 6428 6217 6421 34 26 71 C ATOM 1652 OG SER A 442 20.685 −19.603 −6.297 1.00 50.93 O ANISOU 1652 OG SER A 442 6648 6207 6496 55 −43 19 O ATOM 1653 C SER A 442 22.947 −19.865 −9.145 1.00 51.22 C ANISOU 1653 C SER A 442 6514 6433 6515 40 21 51 C ATOM 1654 O SER A 442 22.689 −20.034 −10.346 1.00 51.69 O ANISOU 1654 O SER A 442 6550 6551 6539 26 −4 45 O ATOM 1655 N LEU A 443 24.012 −20.392 −8.537 1.00 52.28 N ANISOU 1655 N LEU A 443 6616 6628 6619 21 5 55 N ATOM 1656 CA LEU A 443 24.886 −21.388 −9.174 1.00 53.25 C ANISOU 1656 CA LEU A 443 6742 6744 6746 33 0 26 C ATOM 1657 CB LEU A 443 25.940 −21.890 −8.178 1.00 53.57 C ANISOU 1657 CB LEU A 443 6795 6808 6748 31 −4 40 C ATOM 1658 CG LEU A 443 27.061 −22.777 −8.734 1.00 53.94 C ANISOU 1658 CG LEU A 443 6821 6927 6747 71 10 64 C ATOM 1659 CD1 LEU A 443 27.662 −22.226 −10.044 1.00 55.71 C ANISOU 1659 CD1 LEU A 443 7063 7076 7027 −4 40 67 C ATOM 1660 CD2 LEU A 443 28.135 −22.952 −7.702 1.00 55.22 C ANISOU 1660 CD2 LEU A 443 6963 7124 6892 17 6 −2 C ATOM 1661 C LEU A 443 24.112 −22.576 −9.771 1.00 53.71 C ANISOU 1661 C LEU A 443 6819 6793 6792 4 2 27 C ATOM 1662 O LEU A 443 23.550 −23.410 −9.032 1.00 53.76 O ANISOU 1662 O LEU A 443 6815 6847 6764 25 9 84 O ATOM 1663 N SER A 444 24.103 −22.645 −11.106 1.00 54.22 N ANISOU 1663 N SER A 444 6928 6846 6826 −17 15 25 N ATOM 1664 CA SER A 444 23.320 −23.649 −11.835 1.00 54.89 C ANISOU 1664 CA SER A 444 6984 6928 6940 −35 12 13 C ATOM 1665 CB SER A 444 23.149 −23.261 −13.303 1.00 54.86 C ANISOU 1665 CB SER A 444 6962 6941 6939 −49 23 16 C ATOM 1666 OG SER A 444 22.201 −24.103 −13.928 1.00 54.82 O ANISOU 1666 OG SER A 444 7079 6949 6799 −69 14 −88 O ATOM 1667 C SER A 444 23.919 −25.055 −11.752 1.00 55.51 C ANISOU 1667 C SER A 444 7024 6992 7072 −18 33 3 C ATOM 1668 O SER A 444 25.131 −25.226 −11.969 1.00 55.87 O ANISOU 1668 O SER A 444 7048 7066 7112 −44 54 −23 O ATOM 1669 N PRO A 445 23.069 −26.066 −11.448 1.00 55.87 N ANISOU 1669 N PRO A 445 7077 7024 7123 −21 55 28 N ATOM 1670 CA PRO A 445 23.527 −27.447 −11.334 1.00 56.18 C ANISOU 1670 CA PRO A 445 7138 7066 7139 −14 43 20 C ATOM 1671 CB PRO A 445 22.634 −28.013 −10.213 1.00 56.09 C ANISOU 1671 CB PRO A 445 7127 7062 7121 −19 38 43 C ATOM 1672 CG PRO A 445 21.374 −27.115 −10.201 1.00 55.99 C ANISOU 1672 CG PRO A 445 7107 7028 7136 −18 60 17 C ATOM 1673 CD PRO A 445 21.619 −25.973 −11.182 1.00 55.94 C ANISOU 1673 CD PRO A 445 7068 7042 7143 −13 32 27 C ATOM 1674 C PRO A 445 23.313 −28.224 −12.636 1.00 56.28 C ANISOU 1674 C PRO A 445 7162 7119 7100 −20 5 9 C ATOM 1675 O PRO A 445 22.773 −27.671 −13.598 1.00 56.74 O ANISOU 1675 O PRO A 445 7250 7159 7148 −40 −2 46 O ATOM 1676 C1 NAG C 1 23.582 33.784 −6.381 1.00 62.40 C ANISOU 1676 C1 NAG C 1 7883 7954 7870 −44 −8 −30 C ATOM 1677 C2 NAG C 1 23.462 33.722 −7.905 1.00 65.49 C ANISOU 1677 C2 NAG C 1 8282 8330 8269 −28 6 −4 C ATOM 1678 N2 NAG C 1 23.093 35.025 −8.441 1.00 66.24 N ANISOU 1678 N2 NAG C 1 8456 8344 8368 18 29 25 N ATOM 1679 O7 NAG C 1 23.585 35.522 −9.579 1.00 67.45 C ANISOU 1679 C7 NAG C 1 8530 8535 8561 −1 −2 14 C ATOM 1680 O7 NAG C 1 23.964 34.827 −10.522 1.00 67.51 O ANISOU 1680 O7 NAG C 1 8538 8539 8571 59 54 −2 O ATOM 1681 C8 NAG C 1 23.648 37.017 −9.676 1.00 66.89 C ANISOU 1681 C8 NAG C 1 8466 8432 8517 −1 16 31 C ATOM 1682 C3 NAG C 1 22.443 32.665 −8.344 1.00 65.88 C ANISOU 1682 C3 NAG C 1 8333 8317 8379 −25 6 −44 C ATOM 1683 O3 NAG C 1 22.590 32.483 −9.734 1.00 66.07 O ANISOU 1683 O3 NAG C 1 8394 8322 8385 −34 33 −29 O ATOM 1684 C4 NAG C 1 22.599 31.331 −7.598 1.00 65.99 C ANISOU 1684 C4 NAG C 1 8321 8333 8418 5 7 −11 C ATOM 1685 O4 NAG C 1 21.474 30.499 −7.821 1.00 66.44 O ANISOU 1685 O4 NAG C 1 8287 8408 8547 13 31 9 O ATOM 1686 C5 NAG C 1 22.752 31.577 −6.097 1.00 66.41 C ANISOU 1686 C5 NAG C 1 8343 8413 8474 −27 15 −25 C ATOM 1687 C6 NAG C 1 22.991 30.280 −5.319 1.00 70.02 C ANISOU 1687 C6 NAG C 1 8892 8745 8966 21 22 38 C ATOM 1688 O6 NAG C 1 23.519 30.524 −4.020 1.00 73.85 O ANISOU 1688 O6 NAG C 1 9391 9317 9349 −6 −44 −16 O ATOM 1689 O5 NAG C 1 23.817 32.482 −5.871 1.00 63.94 O ANISOU 1689 O5 NAG C 1 8161 8050 8084 80 50 57 O ATOM 1690 C1 NAG C 2 21.730 29.460 −8.798 1.00 65.34 C ANISOU 1690 C1 NAG C 2 8142 8275 8407 44 10 1 C ATOM 1691 C2 NAG C 2 21.096 28.144 −8.338 1.00 65.73 C ANISOU 1691 C2 NAG C 2 8184 8346 8442 35 −16 −3 C ATOM 1692 N2 NAG C 2 21.683 27.718 −7.084 1.00 64.89 N ANISOU 1692 N2 NAG C 2 8185 8149 8320 −5 14 −2 N ATOM 1693 C7 NAG C 2 20.978 27.261 −6.059 1.00 63.76 C ANISOU 1693 C7 NAG C 2 8044 7955 8225 −2 10 −19 C ATOM 1694 O7 NAG C 2 19.755 27.237 −6.045 1.00 64.27 O ANISOU 1694 O7 NAG C 2 8126 7933 8360 58 −45 −18 O ATOM 1695 C8 NAG C 2 21.759 26.753 −4.886 1.00 63.17 C ANISOU 1695 C8 NAG C 2 8009 7902 8089 −43 0 −70 C ATOM 1696 C3 NAG C 2 21.272 27.045 −9.384 1.00 66.30 C ANISOU 1696 C3 NAG C 2 8307 8391 8490 −9 39 −16 C ATOM 1697 O3 NAG C 2 20.552 25.897 −9.004 1.00 66.84 O ANISOU 1697 O3 NAG C 2 8376 8464 8554 36 21 −17 O ATOM 1698 O4 NAG C 2 20.804 27.518 −10.758 1.00 67.01 C ANISOU 1698 C4 NAG C 2 8356 8544 8559 11 6 16 C ATOM 1699 O4 NAG C 2 21.178 26.580 −11.745 1.00 68.38 O ANISOU 1699 O4 NAG C 2 8536 8661 8781 48 1 −50 O ATOM 1700 O5 NAG C 2 21.470 28.857 −11.105 1.00 66.52 C ANISOU 1700 C5 NAG C 2 8316 8433 8524 47 10 −29 C ATOM 1701 O6 NAG C 2 20.959 29.383 −12.447 1.00 65.54 C ANISOU 1701 C6 NAG C 2 8421 8024 8454 24 13 40 C ATOM 1702 O6 NAG C 2 20.896 30.794 −12.448 1.00 66.76 O ANISOU 1702 O6 NAG C 2 8300 8669 8396 −35 74 −46 O ATOM 1703 O5 NAG C 2 21.262 29.813 −10.083 1.00 65.04 O ANISOU 1703 O5 NAG C 2 8097 8260 8355 41 32 14 O ATOM 1704 C1 BMA C 3 20.170 25.602 −12.036 1.00 69.16 C ANISOU 1704 C1 BMA C 3 8600 8769 8908 4 18 7 C ATOM 1705 C2 BMA C 3 20.218 25.313 −13.529 1.00 70.30 C ANISOU 1705 O2 BMA C 3 8850 8920 8938 19 27 −8 C ATOM 1706 O2 BMA C 3 21.569 24.990 −13.866 1.00 70.44 O ANISOU 1706 O2 BMA C 3 8922 8873 8967 −10 1 −5 O ATOM 1707 C3 BMA C 3 19.301 24.146 −13.887 1.00 71.64 C ANISOU 1707 O3 BMA C 3 8983 9046 9190 8 23 33 C ATOM 1708 O3 BMA C 3 19.431 23.763 −15.259 1.00 75.50 O ANISOU 1708 O3 BMA C 3 9547 9570 9568 −21 91 −86 O ATOM 1709 C4 BMA C 3 19.635 22.931 −13.035 1.00 69.90 C ANISOU 1709 C4 BMA C 3 8765 8895 8898 29 63 −28 C ATOM 1710 O4 BMA C 3 18.694 21.909 −13.353 1.00 69.61 O ANISOU 1710 O4 BMA C 3 8791 8790 8869 −4 63 40 O ATOM 1711 C5 BMA C 3 19.565 23.304 −11.562 1.00 68.25 C ANISOU 1711 C5 BMA C 3 8529 8638 8763 43 25 26 C ATOM 1712 C6 BMA C 3 19.992 22.163 −10.656 1.00 66.92 C ANISOU 1712 C6 BMA C 3 8364 8482 8579 9 30 −59 C ATOM 1713 O6 BMA C 3 20.187 22.675 −9.336 1.00 64.81 O ANISOU 1713 O6 BMA C 3 8159 8199 8264 28 −14 −16 O ATOM 1714 O5 BMA C 3 20.426 24.412 −11.292 1.00 68.53 O ANISOU 1714 O5 BMA C 3 8472 8748 8818 3 −2 14 O ATOM 1715 C1 MAN C 4 18.485 24.451 −16.100 1.00 80.48 C ANISOU 1715 C1 MAN C 4 10175 10231 10170 54 −11 39 C ATOM 1716 C2 MAN C 4 17.857 23.471 −17.095 1.00 83.30 C ANISOU 1716 C2 MAN C 4 10595 10536 10515 −34 3 −45 C ATOM 1717 O2 MAN C 4 16.758 24.059 −17.785 1.00 86.66 O ANISOU 1717 O2 MAN C 4 10987 10961 10976 56 −72 11 O ATOM 1718 C3 MAN C 4 18.909 22.950 −18.097 1.00 83.53 C ANISOU 1718 C3 MAN C 4 10571 10594 10570 −3 −6 −16 C ATOM 1719 O3 MAN C 4 18.291 22.361 −19.225 1.00 83.93 O ANISOU 1719 O3 MAN C 4 10617 10659 10610 21 0 −35 O ATOM 1720 C4 MAN C 4 19.916 24.011 −18.569 1.00 83.25 C ANISOU 1720 C4 MAN C 4 10531 10556 10544 0 21 −13 C ATOM 1721 O4 MAN C 4 21.086 23.342 −18.983 1.00 82.77 O ANISOU 1721 O4 MAN C 4 10525 10482 10441 −7 36 −14 O ATOM 1722 C5 MAN C 4 20.281 25.007 −17.461 1.00 82.93 C ANISOU 1722 C5 MAN C 4 10477 10508 10524 23 16 −3 C ATOM 1723 C6 MAN C 4 21.110 26.187 −17.945 1.00 82.33 C ANISOU 1723 C6 MAN C 4 10560 10453 10268 −61 −54 −113 C ATOM 1724 O6 MAN C 4 21.526 26.916 −16.808 1.00 84.80 O ANISOU 1724 O6 MAN C 4 10612 10772 10835 71 57 111 O ATOM 1725 O5 MAN C 4 19.111 25.485 −16.823 1.00 81.69 O ANISOU 1725 O5 MAN C 4 10367 10342 10328 −38 18 6 O ATOM 1726 C1 NAG C 5 15.457 23.728 −17.226 1.00 89.09 C ANISOU 1726 C1 NAG C 5 11237 11320 11293 −16 29 −14 C ATOM 1727 C2 NAG C 5 14.333 24.200 −18.167 1.00 90.32 C ANISOU 1727 O2 NAG C 5 11439 11436 11441 8 −17 15 C ATOM 1728 N2 NAG C 5 13.146 24.596 −17.413 1.00 90.36 N ANISOU 1728 N2 NAG C 5 11463 11426 11444 5 8 −13 N ATOM 1729 C7 NAG C 5 12.233 25.470 −17.860 1.00 91.03 C ANISOU 1729 C7 NAG C 5 11565 11585 11434 −4 21 −26 C ATOM 1730 O7 NAG C 5 12.477 26.652 −18.106 1.00 90.45 O ANISOU 1730 O7 NAG C 5 11527 11441 11396 −12 27 −5 O ATOM 1731 C8 NAG C 5 10.835 24.956 −18.055 1.00 90.77 C ANISOU 1731 C8 NAG C 5 11519 11492 11476 −12 −1 −20 C ATOM 1732 C3 NAG C 5 13.944 23.170 −19.237 1.00 91.25 C ANISOU 1732 C3 NAG C 5 11585 11552 11533 1 −6 −9 C ATOM 1733 O3 NAG C 5 14.135 23.730 −20.517 1.00 91.88 O ANISOU 1733 O3 NAG C 5 11692 11628 11590 −8 8 −1 O ATOM 1734 C4 NAG C 5 14.713 21.851 −19.167 1.00 91.47 C ANISOU 1734 C4 NAG C 5 11616 11584 11554 13 −11 −4 C ATOM 1735 O4 NAG C 5 13.982 20.876 −19.882 1.00 91.95 O ANISOU 1735 O4 NAG C 5 11633 11671 11632 −13 −47 −33 O ATOM 1736 C5 NAG C 5 14.961 21.352 −17.736 1.00 91.25 C ANISOU 1736 C5 NAG C 5 11588 11546 11537 9 −15 −10 C ATOM 1737 C6 NAG C 5 16.052 20.276 −17.726 1.00 92.24 C ANISOU 1737 C6 NAG C 5 11575 11579 11891 0 −46 −137 C ATOM 1738 O6 NAG C 5 16.007 19.560 −16.510 1.00 90.72 O ANISOU 1738 O6 NAG C 5 11735 11388 11344 −24 −54 91 O ATOM 1739 O5 NAG C 5 15.297 22.377 −16.797 1.00 90.15 O ANISOU 1739 O5 NAG C 5 11430 11414 11406 5 −15 26 O ATOM 1740 C1 MAN C 7 20.272 21.548 −8.453 1.00 64.56 C ANISOU 1740 C1 MAN C 7 8141 8188 8199 −9 −4 −47 C ATOM 1741 C2 MAN C 7 19.819 21.886 −7.041 1.00 64.34 C ANISOU 1741 C2 MAN C 7 8131 8140 8175 3 1 −13 C ATOM 1742 O2 MAN C 7 19.851 20.689 −6.290 1.00 64.37 O ANISOU 1742 O2 MAN C 7 8170 8136 8152 13 14 −72 O ATOM 1743 O3 MAN C 7 20.773 22.900 −6.390 1.00 64.46 C ANISOU 1743 C3 MAN C 7 8127 8187 8177 −25 13 −19 C ATOM 1744 O3 MAN C 7 20.410 23.129 −5.037 1.00 63.53 O ANISOU 1744 O3 MAN C 7 7975 8057 8105 −67 −15 −101 O ATOM 1745 O4 MAN C 7 22.239 22.434 −6.476 1.00 64.50 C ANISOU 1745 C4 MAN C 7 8179 8148 8180 7 13 −40 C ATOM 1746 O4 MAN C 7 23.132 23.509 −6.221 1.00 64.35 O ANISOU 1746 O4 MAN C 7 8206 8105 8139 46 −5 −78 O ATOM 1747 O5 MAN C 7 22.602 21.790 −7.827 1.00 64.16 C ANISOU 1747 C5 MAN C 7 8159 8123 8096 −6 0 −11 C ATOM 1748 O6 MAN C 7 23.842 20.915 −7.667 1.00 63.34 C ANISOU 1748 C6 MAN C 7 7929 7985 8151 −24 −13 57 C ATOM 1749 O6 MAN C 7 24.053 20.180 −8.846 1.00 61.06 O ANISOU 1749 O6 MAN C 7 7751 7711 7737 40 −16 −78 O ATOM 1750 O5 MAN C 7 21.565 20.982 −8.380 1.00 64.42 O ANISOU 1750 O5 MAN C 7 8221 8144 8112 −36 17 −13 O ATOM 1751 C1 NAG C 8 18.593 20.003 −6.270 1.00 63.46 C ANISOU 1751 C1 NAG C 8 8070 8012 8030 −5 −1 −34 C ATOM 1752 C2 NAG C 8 18.856 18.592 −5.766 1.00 64.09 C ANISOU 1752 O2 NAG C 8 8169 8111 8072 −6 −10 14 C ATOM 1753 N2 NAG C 8 19.889 17.946 −6.563 1.00 63.96 N ANISOU 1753 N2 NAG C 8 8038 8176 8088 48 −11 12 N ATOM 1754 C7 NAG C 8 21.157 17.831 −6.142 1.00 67.85 C ANISOU 1754 C7 NAG C 8 8475 8217 9086 −90 51 262 C ATOM 1755 O7 NAG C 8 21.540 18.162 −5.017 1.00 63.54 O ANISOU 1755 O7 NAG C 8 8084 8252 7806 −67 −148 −118 O ATOM 1756 C8 NAG C 8 22.133 17.253 −7.129 1.00 63.31 C ANISOU 1756 C8 NAG C 8 8014 8140 7899 131 171 −31 C ATOM 1757 C3 NAG C 8 17.561 17.785 −5.725 1.00 63.80 C ANISOU 1757 C3 NAG C 8 8109 8055 8075 0 −8 −41 C ATOM 1758 O3 NAG C 8 17.802 16.564 −5.040 1.00 64.18 O ANISOU 1758 O3 NAG C 8 8158 8167 8059 20 −18 −53 O ATOM 1759 C4 NAG C 8 16.450 18.562 −4.999 1.00 62.99 C ANISOU 1759 C4 NAG C 8 8022 7934 7977 24 −38 −22 C ATOM 1760 O4 NAG C 8 15.194 17.950 −5.229 1.00 63.13 O ANISOU 1760 O4 NAG C 8 8044 7901 8038 54 −37 −48 O ATOM 1761 C5 NAG C 8 16.391 20.062 −5.340 1.00 62.31 C ANISOU 1761 C5 NAG C 8 7922 7895 7856 3 2 −21 C ATOM 1762 C6 NAG C 8 15.637 20.861 −4.271 1.00 61.45 C ANISOU 1762 C6 NAG C 8 7823 7770 7755 −29 −23 −11 C ATOM 1763 O6 NAG C 8 16.235 20.685 −2.999 1.00 59.23 O ANISOU 1763 O6 NAG C 8 7510 7497 7498 24 71 −106 O ATOM 1764 O5 NAG C 8 17.675 20.637 −5.415 1.00 62.28 O ANISOU 1764 O5 NAG C 8 7944 7849 7870 −26 −26 4 O ATOM 1765 C1 GAL C 9 14.603 17.561 −3.972 1.00 62.63 C ANISOU 1765 C1 GAL C 9 7968 7907 7920 −3 −4 −28 C ATOM 1766 C2 GAL C 9 13.290 16.787 −4.178 1.00 62.38 C ANISOU 1766 C2 GAL C 9 7981 7842 7878 19 −5 −33 C ATOM 1767 O2 GAL C 9 12.274 17.622 −4.709 1.00 60.03 O ANISOU 1767 O2 GAL C 9 7846 7411 7550 −14 6 −131 O ATOM 1768 C3 GAL C 9 12.818 16.148 −2.860 1.00 62.73 C ANISOU 1768 C3 GAL C 9 8016 7945 7872 38 8 −36 C ATOM 1769 O3 GAL C 9 11.849 15.158 −3.102 1.00 62.97 O ANISOU 1769 O3 GAL C 9 7994 7990 7941 −25 39 8 O ATOM 1770 C4 GAL C 9 13.958 15.500 −2.082 1.00 63.15 C ANISOU 1770 C4 GAL C 9 8053 7994 7944 4 6 44 C ATOM 1771 O4 GAL C 9 14.499 14.420 −2.818 1.00 61.84 O ANISOU 1771 O4 GAL C 9 7873 7903 7720 12 9 65 O ATOM 1772 C5 GAL C 9 15.022 16.559 −1.867 1.00 63.69 C ANISOU 1772 C5 GAL C 9 8052 8039 8108 −2 −14 28 C ATOM 1773 O6 GAL C 9 16.117 16.112 −0.903 1.00 64.97 C ANISOU 1773 C6 GAL C 9 8268 8195 8220 44 −44 59 C ATOM 1774 O6 GAL C 9 17.286 16.891 −1.077 1.00 65.24 O ANISOU 1774 O6 GAL C 9 8290 8153 8345 −73 −89 82 O ATOM 1775 O5 GAL C 9 15.525 16.843 −3.157 1.00 64.20 O ANISOU 1775 O5 GAL C 9 8132 8127 8132 15 −28 −36 O ATOM 1776 C1 FUC C 11 24.736 29.847 −3.954 1.00 77.44 C ANISOU 1776 C1 FUC C 11 9759 9799 9837 29 −9 1 C ATOM 1777 O2 FUC C 11 26.080 30.499 −4.293 1.00 78.54 C ANISOU 1777 C2 FUC C 11 9883 9910 10014 −7 4 2 C ATOM 1778 O2 FUC C 11 26.808 30.755 −3.112 1.00 79.77 O ANISOU 1778 O2 FUC C 11 9917 10007 10050 72 −20 −34 O ATOM 1779 O3 FUC C 11 26.977 29.696 −5.231 1.00 78.25 C ANISOU 1779 O3 FUC C 11 9972 9993 10072 −6 44 −18 C ATOM 1780 O3 FUC C 11 26.852 30.264 −6.510 1.00 79.46 O ANISOU 1780 O3 FUC C 11 10023 10064 10081 −17 58 20 O ATOM 1781 C4 FUC C 11 26.769 28.174 −5.286 1.00 77.45 C ANISOU 1781 O4 FUC C 11 10020 10011 10048 −23 33 −20 C ATOM 1782 O4 FUC C 11 26.880 27.703 −6.627 1.00 78.56 O ANISOU 1782 O4 FUC C 11 10076 9967 9991 −28 42 −26 O ATOM 1783 C5 FUC C 11 25.463 27.641 −4.693 1.00 77.17 C ANISOU 1783 O5 FUC C 11 10016 10033 10124 13 23 −2 C ATOM 1784 C6 FUC C 11 24.460 27.240 −5.783 1.00 78.48 C ANISOU 1784 C6 FUC C 11 10059 9937 10067 0 46 −62 C ATOM 1785 O5 FUC C 11 24.873 28.467 −3.681 1.00 79.38 O ANISOU 1785 O5 FUC C 11 9928 9876 10085 −4 13 −44 O ATOM 1786 ZN ZN I 1 1.011 2.625 −6.522 1.00 37.90 ZN ANISOU 1786 ZN ZN I 1 5916 5645 2837 −109 −134 −300 ZN ATOM 1787 ZN ZN I 2 −2.850 29.288 0.411 1.00 66.11 ZN ANISOU 1787 ZN ZN I 2 8310 7792 9014 476 23 −269 ZN ATOM 1788 ZN ZN I 3 0.081 21.125 −18.851 0.50 60.89 ZN ANISOU 1788 ZN ZN I 3 7926 7551 7656 24 −73 15 ZN ATOM 1789 ZN ZN I 4 4.094 −7.924 −14.198 0.50 63.49 ZN ANISOU 1789 ZN ZN I 4 7915 7950 8259 −54 190 −25 ZN ATOM 1790 ON HOH W 1 −2.686 −4.705 −7.680 1.00 51.42 O ANISOU 1790 OW HOH W 1 6584 6695 6258 169 −102 48 O ATOM 1791 ON HOH W 2 15.326 7.920 −11.915 1.00 41.62 O ANISOU 1791 ON HOH W 2 5180 5671 4961 −35 −220 −272 O ATOM 1792 ON HOH W 3 11.705 21.084 −15.919 1.00 53.41 O ANISOU 1792 ON HOH W 3 6696 6842 6755 76 −83 53 O ATOM 1793 ON HOH W 4 4.028 8.613 −6.717 1.00 24.34 O ANISOU 1793 ON HOH W 4 3616 2379 3251 0 −336 632 O ATOM 1794 ON HOH W 5 4.904 7.310 −3.564 1.00 23.00 O ANISOU 1794 ON HOH W 5 3808 3315 1615 −69 307 −476 O ATOM 1795 ON HOH W 6 2.707 2.220 −14.972 1.00 23.50 O ANISOU 1795 ON HOH W 6 3794 2827 2306 91 −630 374 O ATOM 1796 ON HOH W 7 0.086 8.821 −15.891 1.00 13.87 O ANISOU 1796 ON HOH W 7 2598 1403 1268 195 51 −373 O ATOM 1797 ON HOH W 8 23.163 6.265 −13.153 1.00 41.03 O ANISOU 1797 ON HOH W 8 5402 4550 5636 147 229 45 O ATOM 1798 ON HOH W 9 20.619 3.699 −13.114 1.00 23.85 O ANISOU 1798 ON HOH W 9 3742 2526 2794 −240 −69 −539 O ATOM 1799 ON HOH W 10 −2.466 −6.638 −5.878 1.00 44.21 O ANISOU 1799 OW HOH W 10 5803 5719 5275 −213 103 100 O ATOM 1800 OW HOH W 11 12.642 −1.804 −2.353 1.00 63.47 O ANISOU 1800 OW HOH W 11 8214 7905 7995 −35 −45 −9 O ATOM 1801 OW HOH W 12 22.639 6.534 −20.972 1.00 33.47 O ANISOU 1801 OW HOH W 12 3867 4337 4510 −144 129 −236 O ATOM 1802 OW HOH W 13 21.422 1.104 −8.987 1.00 24.16 O ANISOU 1802 OW HOH W 13 3539 3414 2226 −145 221 76 O ATOM 1803 OW HOH W 14 8.879 4.061 −13.080 1.00 24.97 O ANISOU 1803 OW HOH W 14 3170 3239 3075 162 −18 −220 O ATOM 1804 OW HOH W 15 11.288 6.795 −26.054 1.00 45.90 O ANISOU 1804 OW HOH W 15 5829 5389 6220 −13 40 27 O ATOM 1805 OW HOH W 16 14.749 −1.980 −24.051 1.00 16.53 O ANISOU 1805 OW HOH W 16 3271 2274 735 26 224 −273 O ATOM 1806 OW HOH W 17 −0.444 6.851 −20.367 1.00 17.75 O ANISOU 1806 OW HOH W 17 2576 1988 2179 732 234 28 O ATOM 1807 OW HOH W 18 2.245 11.930 −0.120 1.00 32.42 O ANISOU 1807 OW HOH W 18 4359 4213 3746 −160 29 −125 O ATOM 1808 OW HOH W 19 5.162 7.718 −18.328 1.00 25.83 O ANISOU 1808 OW HOH W 19 3497 3439 2878 96 −375 −584 O ATOM 1809 OW HOH W 20 0.796 0.967 −5.140 1.00 21.38 O ANISOU 1809 OW HOH W 20 3124 2654 2344 142 186 −165 O ATOM 1810 OW HOH W 21 −2.715 28.725 2.415 1.00 36.34 O ANISOU 1810 OW HOH W 21 4766 4654 4387 −175 10 163 O ATOM 1811 OW HOH W 22 30.225 −4.400 −9.331 1.00 25.40 O ANISOU 1811 OW HOH W 22 3794 3473 2383 −207 171 921 O ATOM 1812 OW HOH W 23 7.961 6.779 −13.116 1.00 20.66 O ANISOU 1812 OW HOH W 23 2905 2729 2214 −3 224 −281 O ATOM 1813 OW HOH W 24 7.734 8.056 −10.907 1.00 11.86 O ANISOU 1813 OW HOH W 24 2940 1037 527 120 −14 −462 O ATOM 1814 OW HOH W 25 −0.824 −8.657 −5.241 1.00 50.76 O ANISOU 1814 OW HOH W 25 6516 6602 6166 −193 254 42 O ATOM 1815 OW HOH W 26 −5.085 12.307 −13.493 1.00 33.94 O ANISOU 1815 OW HOH W 26 4426 4303 4165 0 −23 −112 O ATOM 1816 OW HOH W 27 21.117 −3.680 −2.105 1.00 36.56 O ANISOU 1816 OW HOH W 27 4582 4997 4310 −135 5 141 O ATOM 1817 OW HOH W 28 26.199 1.780 −6.259 1.00 42.44 O ANISOU 1817 OW HOH W 28 5350 5564 5209 −205 54 −69 O ATOM 1818 OW HOH W 29 25.352 2.736 −9.492 1.00 26.64 O ANISOU 1818 OW HOH W 29 2921 3682 3517 −164 −195 80 O ATOM 1819 OW HOH W 30 2.621 13.373 −12.530 1.00 27.62 O ANISOU 1819 OW HOH W 30 3212 3716 3565 −15 69 381 O ATOM 1820 OW HOH W 31 1.676 −5.459 −13.242 1.00 40.05 O ANISOU 1820 OW HOH W 31 5122 5075 5018 −150 −104 −138 O ATOM 1821 OW HOH W 32 5.616 −7.649 −12.054 1.00 25.11 O ANISOU 1821 OW HOH W 32 3700 2636 3203 249 375 178 O ATOM 1822 OW HOH W 33 0.073 12.268 −18.854 0.50 29.68 O ANISOU 1822 OW HOH W 33 4084 3873 3317 5 87 −2 O ATOM 1823 OW HOH W 34 −0.277 3.231 −8.278 1.00 36.13 O ANISOU 1823 OW HOH W 34 4577 4672 4477 −7 −155 −157 O ATOM 1824 OW HOH W 35 19.204 7.619 −19.539 1.00 41.16 O ANISOU 1824 OW HOH W 35 5228 5167 5241 76 175 −195 O ATOM 1825 OW HOH W 36 21.318 8.586 −18.968 1.00 51.27 O ANISOU 1825 OW HOH W 36 6874 6589 6015 −17 −36 −19 O ATOM 1826 OW HOH W 37 20.898 9.827 −16.899 1.00 40.21 O ANISOU 1826 OW HOH W 37 5588 4786 4901 −99 37 53 O ATOM 1827 OW HOH W 38 19.991 12.076 −17.304 1.00 40.03 O ANISOU 1827 OW HOH W 38 5127 5018 5064 −96 287 75 O ATOM 1828 OW HOH W 39 22.786 6.524 −23.584 1.00 29.46 O ANISOU 1828 OW HOH W 39 4105 3167 3919 −272 63 −315 O ATOM 1829 OW HOH W 40 12.659 7.843 −28.830 1.00 40.36 O ANISOU 1829 OW HOH W 40 5338 4722 5272 −206 −20 69 O ATOM 1830 OW HOH W 41 12.960 24.065 −13.045 1.00 46.62 O ANISOU 1830 OW HOH W 41 5904 6077 5733 92 33 −161 O ATOM 1831 OW HOH W 42 11.135 11.754 −10.185 1.00 30.37 O ANISOU 1831 OW HOH W 42 4271 4163 3103 204 −349 187 O ATOM 1832 OW HOH W 43 13.202 12.031 −11.515 1.00 36.35 O ANISOU 1832 OW HOH W 43 4845 3955 5010 28 −111 −34 O ATOM 1833 OW HOH W 44 10.537 13.629 −2.714 1.00 36.03 O ANISOU 1833 OW HOH W 44 4892 4473 4324 −219 −62 −316 O ATOM 1834 OW HOH W 45 13.983 −0.137 1.252 0.50 26.99 O ANISOU 1834 OW HOH W 45 3481 3502 3270 −79 −42 −87 O ATOM 1835 OW HOH W 46 13.547 11.524 0.333 1.00 50.26 O ANISOU 1835 OW HOH W 46 6295 6703 6097 45 −125 13 O ATOM 1836 OW HOH W 47 −3.193 30.208 −1.659 1.00 52.68 O ANISOU 1836 OW HOH W 47 6778 6676 6562 −56 51 −22 O ATOM 1837 OW HOH W 48 2.590 0.640 −3.975 1.00 38.51 O ANISOU 1837 OW HOH W 48 4885 5274 4470 286 140 −394 O ATOM 1838 OW HOH W 49 −4.829 10.389 −8.743 1.00 30.98 O ANISOU 1838 OW HOH W 49 4328 4157 3286 −6 85 54 O ATOM 1839 OW HOH W 50 −5.682 15.166 −5.147 1.00 62.12 O ANISOU 1839 OW HOH W 50 7854 7808 7937 93 −68 −65 O ATOM 1840 OW HOH W 51 9.256 11.495 −13.968 1.00 24.43 O ANISOU 1840 OW HOH W 51 3144 3093 3042 17 −92 −182 O ATOM 1841 OW HOH W 52 10.348 13.858 −13.762 1.00 31.05 O ANISOU 1841 OW HOH W 52 3599 3978 4219 −257 −125 70 O ATOM 1842 OW HOH W 53 1.049 14.118 −17.122 1.00 31.08 O ANISOU 1842 OW HOH W 53 3973 4342 3492 −11 −363 −196 O ATOM 1843 OW HOH W 54 1.515 10.299 −19.955 1.00 35.99 O ANISOU 1843 OW HOH W 54 4519 4385 4770 160 52 168 O ATOM 1844 OW HOH W 55 2.150 9.713 −17.242 1.00 38.39 O ANISOU 1844 OW HOH W 55 4625 5500 4461 81 −84 35 O ATOM 1845 OW HOH W 56 3.020 6.247 −17.374 1.00 18.01 O ANISOU 1845 OW HOH W 56 3197 2872 774 149 −339 −801 O ATOM 1846 OW HOH W 57 7.330 25.213 −16.752 1.00 49.75 O ANISOU 1846 OW HOH W 57 6292 6287 6321 −124 −65 56 O ATOM 1847 OW HOH W 58 6.140 4.420 −24.421 1.00 31.76 O ANISOU 1847 OW HOH W 58 4147 4290 3628 107 5 22 O ATOM 1848 OW HOH W 59 6.934 6.415 −25.965 1.00 48.06 O ANISOU 1848 OW HOH W 59 6126 6215 5917 104 −184 125 O ATOM 1849 OW HOH W 60 2.789 −10.262 −14.281 1.00 52.27 O ANISOU 1849 OW HOH W 60 6829 6640 6389 −85 61 −132 O ATOM 1850 OW HOH W 61 10.173 −11.474 −17.131 1.00 43.89 O ANISOU 1850 OW HOH W 61 5243 5991 5441 −19 145 −51 O ATOM 1851 OW HOH W 62 10.423 −9.588 −15.301 1.00 41.60 O ANISOU 1851 OW HOH W 62 4910 5490 5403 −299 −111 149 O ATOM 1852 OW HOH W 63 12.562 10.469 −16.760 1.00 47.26 O ANISOU 1852 OW HOH W 63 6061 6168 5726 54 −119 90 O ATOM 1853 OW HOH W 64 25.207 4.876 −13.048 1.00 43.98 O ANISOU 1853 OW HOH W 64 5382 5861 5465 4 11 −150 O ATOM 1854 OW HOH W 65 20.403 6.945 −24.246 1.00 31.68 O ANISOU 1854 OW HOH W 65 4456 4465 3114 −263 −194 −229 O ATOM 1855 OW HOH W 66 16.219 5.833 −30.699 1.00 53.61 O ANISOU 1855 OW HOH W 66 6744 6870 6754 −31 −25 −174 O ATOM 1856 OW HOH W 67 15.439 −3.579 −28.825 1.00 50.26 O ANISOU 1856 OW HOH W 67 6356 6649 6091 19 −89 −6 O ATOM 1857 OW HOH W 68 0.448 −2.007 −9.703 1.00 47.76 O ANISOU 1857 OW HOH W 68 5769 6029 6348 −118 −41 33 O ATOM 1858 OW HOH W 69 9.088 10.333 −11.458 1.00 45.89 O ANISOU 1858 OW HOH W 69 5637 5868 5931 55 174 101 O ATOM 1859 OW HOH W 70 2.301 8.833 3.077 1.00 41.79 O ANISOU 1859 OW HOH W 70 5409 5255 5212 66 −371 −158 O ATOM 1860 OW HOH W 71 19.184 33.621 −4.885 1.00 59.40 O ANISOU 1860 OW HOH W 71 7601 7341 7625 −69 −45 −52 O ATOM 1861 OW HOH W 72 11.411 47.589 −7.153 1.00 50.72 O ANISOU 1861 OW HOH W 72 6739 6215 6314 −100 −123 −98 O ATOM 1862 OW HOH W 73 −2.721 22.353 −19.060 1.00 50.88 O ANISOU 1862 OW HOH W 73 6801 6612 5916 38 18 −78 O ATOM 1863 OW HOH W 74 −2.520 30.795 −12.887 1.00 44.98 O ANISOU 1863 OW HOH W 74 5923 5326 5838 −14 57 136 O ATOM 1864 OW HOH W 75 35.811 −10.347 −12.530 1.00 32.21 O ANISOU 1864 OW HOH W 75 4854 4402 2979 196 130 6 O END

Conclusion:

The three-dimensional structure of Fc/TM was found to be very similar to that of other unliganded, unmutated human Fc regions. The dramatic, broad-ranging functional effects of the TM set of substitutions were not caused by major structural rearrangements in the Fc structure, but rather by the localized loss of a few interactions at the mutation sites.

6.34 Example 34: Internalization of Anti-IFNAR1 Antibodies

Purpose:

To investigate the ability of anti-IFNAR1 antibodies to internalize in cells.

Methods:

THP-1 cells were cultured in RPMI-1640 media containing 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum at 37° C. in 5% CO₂ incubator. THP-1 cells were seeded at 2×10⁵ cells/ml in fresh growth media one day prior to experiments. At the day of the experiment, cells were washed, counted and resuspended in PBS at 3×10⁶ cells/ml. The cells were stained with 1 μM CFSE in 37° C. CO₂ incubator for 10 min. Following additional two washes with PBS, the cells were placed on ice and incubated with FcR block using 20 μl per 10⁶ cells on ice for 5 min and then stained with 1 μg/ml of Alexa647-9D4-TM or Alexa 647-R347 (non-specific control antibody) on ice for 1 h. After removal of unbound mAb by 3 washes with PBS, cells were resuspended in PBS containing 2% BSA and sodium azide. The internalization was initiated by transferring the cells to an environmentally controlled chamber under 37° C., 5% CO₂ and 70% humidity and the internalization kinetics of Alexa647-9D4-TM was recorded over time by imaging the fluorescence of cells.

The fluorescence images of cells were analyzed using an algorithm. The algorithm used CFSE cytosolic dye to identify the boundary of a cell and a membrane region. The algorithm quantified the 9D4-TM associated fluorescence inside cells as well as on membrane. Rate of fluorescence accumulated inside the cells was calculated by model fitting of the data using SAAMII software.

Results:

Alexa647-9D4-TM bound to THP-1 cells. No binding of Alexa647-R347, the isotype control of 9D4-TM, was observed on the same cells. This result demonstrated specific binding to THP-1 cells by 9D4-TM (FIG. 33). At 4° C., 9D4-TM binding was predominately located at cell surface (0 min—FIG. 33). Once the cells were incubated at 37° C., the fluorescence signal for 9D4-TM staining was significantly decreased from cell surface and accumulated in cytosolic compartment as punctuated spots. Kinetic images recorded over 60 min indicated gradual migration of fluorescence from cell surface to punctuated spots located at cytosolic compartment (15, 30 and 50 min time points, FIG. 33). The result clearly demonstrated internalization of 9D4-TM on THP-1 cells.

6.35 Example 35: Absence of 9D4-TM Mediated CDC Activity

Purpose:

To determine if 9D4-TM is unable to induce CDC activity a series of experiments were conducted.

Methods:

Freshly isolated human blood from healthy, human donors was collected (approximately 100 ml) and spun down for 10 minutes at 3000G to separate serum from cells. The serum fraction was separated into two tubes. The first tube was diluted with phenol-free RPMI 1640 to a final concentration of 10% serum (non-heat inactivated or NHI). The second tube was placed in a 56° C. water bath for 30 minutes to heat inactivate the complement components. Subsequently, the second tube was diluted with phenol-free RPMI-1640 media to a final concentration of 10% heat-inactivated (HI) human serum.

Daudi B cells were used as target cells as they express CD20 (target for positive control antibody) and IFNAR1. Target cells were washed and resuspended in either phenol-free RPMI media with 10% non-heat inactivated serum or in phenol-free RPMI media with 10% heat inactivated serum at a final concentration of 0.4×10⁶ cells/mL. Antibody solutions were prepared as a 3× dilution series with the concentrations ranging from 50 ug/mL-1.3×10⁻⁶ μg/mL. Replicate preparations of antibody dilutions were made in either media with heat-inactivated or non-heat-inactivated human serum. The CDC assay was prepared by adding 50 μL of NHI or HI media to appropriate wells of a 96 well, round bottom plate. 50 μL of antibody dilution series were added to the appropriate wells. Subsequently, 50 μL of the target cell preparation was added to the wells, including extra wells with target cells alone for controls. The plates were incubated for 37° C. for 4 hours in 5% CO₂. After 3.5 hour incubation, 20 uL lysis buffer was added to appropriate control wells designated for determination of maximum lysis signal. The Quantitate™ LDH release assay was performed using protocols defined in Promega non-radioactive cytotoxicity assay, #G1780. Absorbance was measured at 490 nM and Kd values were generated using GraphPad Prism 4 analysis software.

Results:

Presented in FIG. 34 are the results from the CDC performed as described above. The modified anti-IFNAR1 antibody, 9D4-TM exhibited no detectable CDC activity on target Daudi B cells over that observed with the R347 antibody. In contrast, the positive control antibody, which binds CD20 expressed on Daudi B cells, caused a dose-dependent increase in cytotoxicity over background levels. These results confirm that 9D4-TM cannot mediate CDC on IFNAR1 expressing target cells.

6.36 Example 36: The Modified Anti-IFNAR1 Antibody, 9D4-TM does not Display any Adverse Toxicity

Purpose:

To establish that 9D4-TM does not elicit any adverse toxicity, a single-dose toxicity study was performed in cynomolgus monkeys.

Methods:

In this study, 4 groups of 10 animals each (5/sex/group) received a single dose of 0, 5, 30, or 100 mg/kg of 9D4-TM on Day 1. After dosing, 2 animals/sex/group were assigned to necropsy on Day 3 with all remaining animals monitored until Day 70 and then removed from study without necropsy. Toxicity was assessed based on mortality, clinical signs (including menses), immunophenotyping, body weights, physical examinations (including heart rate, respiration rate, and body temperature), clinical pathology, organ weights, and microscopic data.

Results:

Under the conditions outlined above, there were no 9D4-TM-related adverse changes in mortality, clinical signs (including menses), body weight, physical examinations (heart rate, respiration rate and body temperature), clinical pathology, organ weights and microscopic data. These results suggest that the modified anti-IFNAR1 antibody, 9D4-TM does not elicity any adverse toxicity.

Whereas, particular embodiments of the invention have been described above for purposes of description, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. 

We claim:
 1. A modified IgG1 class human monoclonal antibody specific for IFNAR1, wherein said antibody comprises: (i) a human heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; (ii) a human light chain variable region comprising the amino acid sequence of SEQ ID NO: 40; (iii) a human light chain constant region comprising the amino acid sequence of SEQ ID NO: 41; (iv) a human heavy chain constant region comprising the amino acid sequence of SEQ ID NO:
 42. 2. A pharmaceutical composition comprising the antibody of any of the claim 1, and a pharmaceutically acceptable excipient. 